Array and method for detecting spatial information of nucleic acids

ABSTRACT

Provided are a method for detecting spatial information of nucleic acids in a sample, as well as a nucleic acid array used in the method and a method for producing the nucleic acid array.

TECHNICAL FIELD

The invention relates to the field of biomolecule spatial detection.Specifically, the present invention provides a method for detectingspatial information of nucleic acid in a sample, a nucleic acid arrayused in the method, and a method for producing the nucleic acid array.

BACKGROUND ART

The spatial locations of cells in a tissue significantly affects theirfunctions. In order to explore this spatial heterogeneity, it isnecessary to quantify and analyze the cell's genome or transcriptomewith the knowledge of the spatial coordinates. However, collecting smalltissue regions or even single cells for genome or transcriptome analysisis very laborious, costly, and with low precision. Therefore, it is verynecessary to develop a method that can achieve single-cellular or evensubcellular level and high-throughput detection of spatial information(for example, nucleic acid location, distribution, and/or expression) ofa biomolecule (for example, a nucleic acid).

CONTENTS OF THE INVENTION

In order to realize the spatial detection of nucleic acid, the prior artcombines array technology with high-throughput DNA sequencing technologyto capture and label nucleic acid with positional tag in a tissuesample, and sequence and analyze it. In order to obtain a chip that canachieve the above-mentioned purpose, the prior art fixes a probe capableof capturing nucleic acid on the chip by spotting or a bead-basedmethod. However, the active region size of the chip obtained by usingthe micro-volume spotting system for the method of droplet spotting on aplane is as high as 200 microns, and the cell observation precision isonly 20 cells; the active region size of the chip obtained by using thebead-based method with spread-plating of beads labelled with positionaltags is up to 10 microns, and the cell observation precision can onlyreach a single cell level, and the subcellular level cannot be achieved.The present invention provides a novel nucleic acid array for nucleicacid spatial detection, a preparation method thereof, and a nucleic acidspatial detection method based on the array, which can simultaneouslyrealize high-precision subcellular localization and high-throughputtissue localization, and has important applications value.

Preparation of Nucleic Acid Array

In the first aspect, the present invention provides a method forgenerating a nucleic acid array for detecting spatial information of anucleic acid in a biological sample, the method comprising the followingsteps:

(1) providing multiple kinds of carrier sequences, each kind of carriersequence comprises a plurality of copies of the carrier sequence, andthe carrier sequence in the direction from 5′ to 3′ comprises apositioning sequence and a first immobilization sequence,

the positioning sequence has a unique nucleotide sequence correspondingto the position of the kind of carrier sequence on the array;

the first immobilization sequence allows annealing to its complementarynucleotide sequence and initiating an extension reaction;

(2) ligating the multiple kinds of carrier sequences to a surface of asolid support (e.g., a chip);

(3) providing a first primer, and using the carrier sequence as atemplate to perform a primer extension reaction, so that the region ofthe first immobilization sequence and the positioning sequence of thecarrier sequence forms a double strand, wherein the strand thathybridizes with the carrier sequence is a first nucleic acid molecule,the first nucleic acid molecule in the direction from 5′ to 3′ comprisesa complementary sequence of the first immobilization sequence and thepositioning sequence; wherein, the first primer at its 3′ end comprisesa first immobilization sequence complementary region, the firstimmobilization sequence complementary region comprises a complementarysequence of the first immobilization sequence or a fragment thereof, andhas a free 3′ end.

In certain embodiments, the carrier sequence and the first nucleic acidmolecule are single-stranded nucleic acid sequences. In someembodiments, the carrier sequence and the first nucleic acid moleculeare single-stranded DNA sequences.

In some embodiments, in step (3), while performing the extensionreaction, the carrier sequence is sequenced, so as to obtain thesequence information of the positioning sequence contained in thecarrier sequence.

In some embodiments, in step (1), the multiple kinds of carriersequences are provided through the following steps:

(i) providing multiple kinds of carrier sequence templates, the carriersequence template comprises the complementary sequence of the carriersequence;

(ii) using each kind of carrier sequence template as a template toperform a nucleic acid amplification reaction so as to obtain anamplification product of each kind of carrier sequence template, inwhich the amplification product comprises a plurality of copies of thecarrier sequence.

In certain embodiments, the amplification is selected from rollingcircle amplification (RCA), bridge PCR amplification, multiple stranddisplacement amplification (MDA), or emulsion PCR amplification.

In certain embodiments, the rolling circle amplification is performed toobtain a DNB formed by a concatemer of the carrier sequence. In suchembodiments, a circular template sequence is provided in step (i). Themethod for preparing circular nucleic acid molecules is a conventionalmethod in the art, and can be selected according to needs by thoseskilled in the art. For example, a linear nucleic acid template can beobtained first, and then circularization of the linear nucleic acidtemplate can be realized by a ligase (e.g., DNA ligase).

In some embodiments, the bridge PCR amplification, emulsion PCRamplification, or multiple strand displacement amplification isperformed to obtain a DNA cluster formed by a clone population of thecarrier sequence.

In some embodiments, the method further comprises the following steps:

(4) providing a second nucleic acid molecule, the second nucleic acidmolecule comprises a capture sequence;

the capture sequence is capable of hybridizing with the whole or part ofa nucleic acid to be captured, and comprises: (a) an oligonucleotidesequence capable of capturing mRNA; and/or, (b) a random or degenerateoligonucleotide sequence; or, (c) a specific sequence for a specifictarget nucleic acid; and, the capture sequence has a free 3′ end toenable the second nucleic acid molecule to function as an extensionprimer,

(5) ligating the second nucleic acid molecule to the first nucleic acidmolecule (for example, ligating the second nucleic acid molecule to thefirst nucleic acid molecule by using a ligase).

In certain embodiments, the second nucleic acid molecule is asingle-stranded nucleic acid sequence. In certain embodiments, thesecond nucleic acid molecule is a single-stranded DNA sequence. Incertain embodiments, the second nucleic acid molecule is asingle-stranded RNA sequence.

In certain embodiments, the second nucleic acid molecule in thedirection from 5′ to 3′ comprises an immobilization region and a capturesequence, and the immobilization region comprises a double-strandednucleic acid sequence, such as a double-stranded DNA sequence. In someembodiments, the capture sequence contained in the second nucleic acidmolecule is a single-stranded nucleic acid sequence, such as asingle-stranded DNA sequence or a single-stranded RNA sequence. It iseasy to understand that in such embodiments, the second nucleic acidmolecule has a partially double-stranded structure, that is, itsimmobilization region has a double-stranded structure, and its capturesequence has a single-stranded structure.

In certain embodiments, the double-stranded nucleic acid sequence has alength of 1 bp to 50 bp, for example, 10 bp to 50 bp, 10 bp to 40 bp, or10 bp to 30 bp.

In other embodiments, the method further comprises the following steps:

(4) providing a second nucleic acid molecule, in which the secondnucleic acid molecule in the direction from 5′ to 3′ comprises acomplement of second immobilization sequence and a capture sequence;

the complement of second immobilization sequence allows hybridizing toits complementary nucleotide sequence;

the capture sequence is capable of hybridizing with the whole or part ofa nucleic acid to be captured, and comprises: (a) an oligonucleotidesequence capable of capturing mRNA; and/or, (b) a random or degenerateoligonucleotide sequence; or, (c) a specific sequence for a specifictarget nucleic acid; and, the capture sequence has a free 3′ end toenable the second nucleic acid molecule to function as an extensionprimer;

(5) hybridizing the complement of second immobilization sequence withthe second immobilization sequence under a condition that allowsannealing, thereby ligating the second nucleic acid molecule to thecarrier sequence;

(6) optionally, ligating the first nucleic acid molecule to the secondnucleic acid molecule that are hybridized to the carrier sequencerespectively (for example, ligating the second nucleic acid molecule tothe first nucleic acid molecule by using a ligase).

In such embodiments, each carrier sequence further comprises a secondimmobilization sequence at its 5′ end, the second immobilizationsequence allows annealing to its complementary nucleotide sequence. Insome embodiments, the second immobilization sequence allows annealing toits complementary nucleotide sequence and initiating an extensionreaction (for example, it can be used as a binding site of a bridge PCRprimer).

In certain embodiments, the second nucleic acid molecule is asingle-stranded nucleic acid sequence. In certain embodiments, thesecond nucleic acid molecule is a single-stranded DNA sequence. Incertain embodiments, the second nucleic acid molecule is asingle-stranded RNA sequence.

In certain embodiments, the second immobilization sequence is adjacentto the positioning sequence.

In some embodiments, the second immobilization sequence has a length of1 bp to 50 bp, for example, 10 bp to 50 bp, 10 bp to 40 bp, 10 bp to 30bp, or 10 bp to 20 bp.

In some embodiments, in step (3), the first primer further comprises aunique molecular identifier (UMI) sequence at the 5′ end of the firstimmobilization sequence complementary region contained therein, so thatthe first nucleic acid molecule comprises a UMI sequence at the 5′ endof the complement of first immobilization sequence contained therein;or, in step (4), the second nucleic acid molecule further comprises aUMI sequence, and the UMI sequence is located at the 5′ end of thecapture sequence;

the UMI sequence is a nucleotide sequence composed of at least 1 (forexample, at least 2, at least 3, at least 4, or at least 5; for example,5 to 100, 5 to 50, 5 to 20, such as 10) nucleotide N, and each N isindependently any one of A, C, G and T.

In some embodiments, when the first primer comprises the uniquemolecular identifier (UMI) sequence at the 5′ end of the firstimmobilization sequence complementary region contained therein, thefirst primer may further comprise an additional sequence at the 5′ endof the UMI sequence.

In some embodiments, the oligonucleotide sequence capable of capturingmRNA comprises a sequence capable of hybridizing with a poly-A tail ofthe mRNA. In certain embodiments, the oligonucleotide sequence capableof capturing mRNA comprises a poly-T oligonucleotide sequence. Incertain embodiments, the poly-T oligonucleotide sequence comprises atleast 10 (e.g., at least 20) deoxythymidine residues.

In certain embodiments, the solid support is a chip. In someembodiments, the solid support can be used as a sequencing platform,such as a sequencing chip. In some embodiments, the solid support is ahigh-throughput sequencing chip, such as a high-throughput sequencingchip used in Illumina, MGI, or Thermo Fisher sequencing platform.

In a second aspect, the present invention provides a method forgenerating a nucleic acid array for detecting spatial information of anucleic acid in a biological sample, the method comprising the followingsteps:

(1) providing multiple kinds of carrier sequences, each kind of carriersequence comprises a plurality of copies of the carrier sequence, thecarrier sequence in the direction from 5′ to 3′ comprises: a capturesequence template, a positioning sequence and a first immobilizationsequence,

the capture sequence template comprises a complementary sequence of acapture sequence, and the capture sequence is capable of hybridizingwith the whole or part of a nucleic acid to be captured, whichcomprises: (a) an oligonucleotide sequence capable of capturing mRNA;and/or, (b) a random or degenerate oligonucleotide sequence; or, (c) aspecific sequence for a specific target nucleic acid;

the positioning sequence has a unique nucleotide sequence correspondingto the position of the kind of carrier sequence on the array;

the first immobilization sequence allows annealing to its complementarynucleotide sequence and initiating an extension reaction; and, the firstimmobilization sequence also comprises a cleavage site, and the cleavagemay be selected from enzymatic cleavage with nicking enzyme, enzymaticcleavage with USER enzyme, photocleavage, chemical cleavage orCRISPR-based cleavage;

(2) ligating the multiple kinds of carrier sequences to a surface of asolid support (e.g., a chip);

(3) providing a first primer (or referred to as a probe primer), thefirst primer in the direction from 5′ to 3′ comprises a binding region,a cleavage region and a first immobilization sequence complementaryregion, and the first immobilization sequence complementary regioncomprises a complementary sequence of the first immobilization sequenceor a fragment thereof, and has a free 3′ end; the binding regioncomprises a linker that can be ligated to the surface of the solidsupport; the cleavage region comprises a cleavage site;

using the first primer as a primer and the carrier sequence as atemplate to perform a primer extension reaction, so that the region ofthe first immobilization sequence, the positioning sequence and thecapture sequence template of the carrier sequence forms adouble-stranded strand, wherein a strand hybridized with the carriersequence is a first nucleic acid molecule, and the first nucleic acidmolecule in the direction from 5′ to 3′ comprises a complement of firstimmobilization sequence, a complement of positioning sequence, and acapture sequence; the first nucleic acid molecule can also be referredto as a capture probe;

(4) ligating the first primer to the surface of the solid support;wherein, steps (3) and (4) are performed in any order;

(5) optionally performing cleavage at the cleavage site contained in thefirst immobilization sequence of the carrier sequence to digest thecarrier sequence, so that the extension product of step (3) is separatedfrom the template (i.e., the carrier sequence) for forming the extensionproduct, so that the first nucleic acid molecule (the capture probe) isligated to the surface of the solid support (e.g., chip).

Since the capture probe (i.e., the first nucleic acid molecule) isobtained by using the carrier sequence as template and performing primerextension, the capture probe comprises a complement of the uniquepositioning sequence corresponding to the position of the kind ofcapture probe (the kind of carrier sequence) on the array, and a capturesequence that can hybridize with the whole or part of a nucleic acidmolecule to be captured. The position of the kind of capture probe onthe array can be determined by analyzing the complement of thepositioning sequence.

In this context, the expression “each kind of carrier sequence” refersto carrier sequences comprising the same positioning sequence.

In some embodiments, in step (1), the multiple kinds of carriersequences are provided through the following steps:

(i) providing multiple kinds of carrier sequence templates, the carriersequence template comprises a complementary sequence of the carriersequence;

(ii) using each kind of carrier sequence template as a template toperform a nucleic acid amplification reaction so as to obtain anamplification product of each kind of carrier sequence template, inwhich the amplification product comprises a plurality of copies of thecarrier sequence.

In certain embodiments, the amplification is selected from rollingcircle amplification (RCA), bridge PCR amplification, multiple stranddisplacement amplification (MDA), or emulsion PCR amplification.

In certain embodiments, the rolling circle amplification is performed toobtain a DNB formed by a concatemer of the carrier sequence. In suchembodiments, a circular template sequence is provided in step (i). Themethod for preparing circular nucleic acid molecules is a conventionalmethod in the art, and can be selected according to needs by thoseskilled in the art. For example, a linear nucleic acid template can beobtained first, and then circularization of the linear nucleic acidtemplate can be realized by a ligase (e.g., a DNA ligase).

In certain embodiments, each kind of carrier sequence is a DNB formed bya concatemer of a plurality of copies of the carrier sequence.

In some embodiments, step (1) comprises the following steps:

(1a) providing a circular nucleic acid template, the circular nucleicacid template comprises one kind of carrier sequence template, thecarrier sequence template comprises a complementary sequence of thecarrier sequence, that is, the carrier sequence template in thedirection from 5′ to 3′ comprises a complement of first immobilizationsequence, a complement of positioning sequence and a capture sequence;

(1b) performing rolling circle amplification (RCA) by using the circularnucleic acid template as a template to obtain a DNA nanoball (DNB)formed by a concatemer of the carrier sequence.

In some embodiments, bridge PCR amplification, emulsion PCRamplification, or multiple strand displacement amplification isperformed to obtain a DNA cluster formed by a clone population of thecarrier sequence.

In some embodiments, the cleavage site contained in the firstimmobilization sequence is a cleavage site of nicking enzyme. In someembodiments, the nicking enzyme is selected from USER, BamHI, BmtI, etc.In certain exemplary embodiments, the cleavage site is shown in SEQ IDNO: 14.

In some embodiments, the first immobilization sequence further comprisesa hybridization region for sequencing primer and/or a hybridizationregion for amplification primer; wherein the hybridization region forsequencing primer allows annealing to a sequencing primer and initiatinga sequencing reaction, and the hybridization region for amplificationprimer allows annealing to an amplification primer and initiating anextension and amplification reaction.

In some embodiments, the first immobilization sequence has a length ofgreater than 1 bp, such as greater than 10 bp, or greater than 20 bp. Insome embodiments, the first immobilization sequence has a length of 20to 100 bp, such as 20 to 80 bp.

In some embodiments, the positioning sequence has a length of greaterthan 1 bp, such as greater than 10 bp. In some embodiments, thepositioning sequence has a length of 10 to 100 bp, such as 10 to 50 bp,such as 10 to 30 bp, such as 20 bp.

In some embodiments, the oligonucleotide sequence capable of capturingmRNA comprises a sequence capable of hybridizing with a poly-A tail ofthe mRNA. In certain embodiments, the oligonucleotide sequence capableof capturing mRNA comprises a poly-T oligonucleotide sequence. Incertain embodiments, the poly-T oligonucleotide sequence comprises atleast 10 (e.g., at least 20) deoxythymidine residues.

In some embodiments, the capture sequence has a length of greater than 1bp. In some embodiments, the capture sequence has a length of 1 to 100bp, such as 10 to 50 bp, such as 10 to 30 bp.

In some embodiments, the carrier sequence further comprises a complementof UMI sequence (also referred to as a probe tag region) locateddownstream of the capture sequence template and upstream of the firstimmobilization sequence, the complement of UMI sequence is complementaryto a UMI sequence, and the UMI sequence is a nucleotide sequencecomposed of at least 1 (for example, at least 2, at least 3, at least 4,or at least 5; for example, 5 to 100, 5 to 50, 5 to 20, such as 10)nucleotide N, wherein each N is independently any one of A, C, G and T.In some embodiments, the complement of UMI sequence is located betweenthe positioning sequence and the capture sequence template. In otherembodiments, the complement of UMI sequence is located between the firstimmobilization sequence and the positioning sequence. In suchembodiments, in step (3), when the primer extension reaction isperformed using the carrier sequence as a template, the first nucleicacid molecule/capture probe that hybridizes to the carrier sequence willcorrespondingly comprise the UMI sequence (also referred to as probetag).

In some embodiments, in order to obtain the aforementioned UMI sequenceor its complementary sequence, a template sequence of the carriersequence (i.e., a carrier sequence template) comprises a UMI sequencetemplate at a corresponding position, and the UMI sequence template is asequence composed of modified bases, the modified bases are capable ofcomplementary pairing by hydrogen bonds with a variety of main bases(for example, C, G, A, T, U); for example, the modified base can beInosine, which is capable of complementary pairing with bases A, C andU. Without being bound by any theory, it is believed that when thecarrier sequence template comprises the UMI sequence template, everytime the amplification is performed in the rolling circle amplificationprocess, the bases capable of complementary pairing with the UMIsequence template are randomly bonded, so that the amplification productof each time has a unique UMI sequence which is randomly formed, therebydistinguishing the amplification product of each time. Thus, forexample, copy number can be quantified for different nucleic acidmolecules which are captured. In some embodiments, the UMI sequencetemplate comprises a plurality of (e.g., at least 10, such as 10 to 100)Inosines. In some embodiments, the UMI sequence template has a length ofgreater than 1 bp. In some embodiments, the UMI sequence template has alength of greater than 5 bp. In some embodiments, the UMI sequencetemplate has a length of 5 to 100 bp, such as 5 to 50 bp, such as 5 to20 bp, such as 5 to 15 bp, such as 10 bp.

In certain embodiments, the solid support is a chip. In someembodiments, the solid support can be used as a sequencing platform. Insome embodiments, the solid support is a sequencing chip (MGI), such asa sequencing chip of BGISEQ-500 platform. In some embodiments, the solidsupport is a high-density array chip, which can be obtained, forexample, by the method described in patent CN103180496B.

The carrier sequence (e.g., DNB) can be ligated to the surface of thesolid support by any suitable method known in the art. In certainembodiments, non-limiting examples of the method include nucleic acidhybridization, biotin-streptavidin binding, sulfhydryl binding,photo-activated binding, covalent binding, antibody-antigen, physicallimitation by hydrogel or other porous polymer materials, etc., or anycombination thereof.

In some embodiments, the solid support is selected from the followingmaterials: glass, silicon, polylysine coating material, nitrocellulose,polystyrene, cyclic olefin copolymers (COCs), cyclic olefins polymers(COPs), polypropylene, polyethylene or polycarbonate, etc.

In some embodiments, in step (3), while performing the primer extensionreaction, the carrier sequence (for example, the positioning sequencecontained therein) is sequenced, so as to obtain the sequenceinformation of the positioning sequence contained in the carriersequence.

In some embodiments, before step (3), a step of sequencing the carriersequence (for example, the positioning sequence contained therein) isfurther comprised. In some embodiments, after the sequencing iscompleted, washing is performed to remove dNTP which has been added tothe synthetic strand due to the sequencing.

In certain embodiments, the linker is a linking group capable ofcoupling with an activating group (e.g., NH₂). In such embodiments, thesurface of the solid support is modified with an activating group (e.g.,NH₂). In some embodiments, the linker comprises —SH, -DBCO, —NHS, andthe like. In certain exemplary embodiments, the linker is DBCO, andAzido-dPEG®8-NHS ester is attached to the surface of the solid support.

In some embodiments, the cleavage site contained in the cleavage regionof the first primer is a site where a controlled cleavage can beperformed by a chemical, enzymatic, or photochemical method. In certainembodiments, the cleavage site is a cleavage site of enzyme. In someembodiments, the enzyme site is an enzyme site of USER enzyme (UUU).

In some embodiments, the cleavage region of the first primer isdifferent from the cleavage site contained in the first immobilizationsequence of the carrier sequence.

In certain embodiments, the amplification comprises PCR.

Nucleic Acid Array and Kit

In a third aspect, the present invention provides a nucleic acid arrayfor detecting spatial information of a nucleic acid in a sample, whichcomprises a solid support (e.g., a chip) with multiple kinds of carriersequences attached to its surface, in which each kind of carriersequence occupies a different position in the array, each kind ofcarrier sequence comprises a plurality of copies of the carriersequence, and the carrier sequence in the direction from 5′ to 3′comprises a positioning sequence and a first immobilization sequence,

the positioning sequence has a unique nucleotide sequence correspondingto the position of the kind of carrier sequence on the array;

the first immobilization sequence allows annealing to its complementarynucleotide sequence and initiating an extension reaction.

In certain embodiments, said each kind of carrier sequence (i.e., thecarrier sequences comprising the same positioning sequence) occupies anarea (i.e., active region) having a diameter of less than 1 micron, forexample, about 900 nanometers, about 800 nanometers, about 700nanometers, about 600 nanometers, or about 500 nanometers, on thesurface of the solid support.

In some embodiments, the nucleic acid array further comprises a firstnucleic acid molecule, in which the first nucleic acid molecule in thedirection from 5′ to 3′ comprises: a complement of first immobilizationsequence and a complement of positioning sequence, and forms a doublestrand structure by hybridizing with the first immobilization sequenceand the positioning sequence of the carrier sequence. It is easy tounderstand that in the first nucleic acid molecule, only the complementof first immobilization sequence and the complement of positioningsequence are complementary to the corresponding sequences of the carriersequence and therefore form a double strand, so that the double strandformed by the first immobilization sequence and the carrier sequence isan incomplete double strand, that is, a partial double-strandedstructure.

In certain embodiments, each copy of each kind of carrier sequencecomprises a first nucleic acid molecule hybridized therewith.

In certain embodiments, the carrier sequence and the first nucleic acidmolecule are single-stranded nucleic acid sequences. In someembodiments, the carrier sequence and the first nucleic acid moleculeare single-stranded DNA sequences.

In some embodiments, the nucleic acid array further comprises a secondnucleic acid molecule, in which the second nucleic acid molecule isligated to the first nucleic acid molecule thereby being immobilized tothe nucleic acid array, and the second nucleic acid molecule comprises acapture sequence;

the capture sequence can hybridize with the whole or part of a nucleicacid to be captured, and comprises: (a) an oligonucleotide sequencecapable of capturing mRNA; and/or, (b) a random or degenerateoligonucleotide sequence; or, (c) a specific sequence for a specifictarget nucleic acid; and, the capture sequence has a free 3′ end toenable the second nucleic acid molecule to function as an extensionprimer.

In certain embodiments, each first nucleic acid molecule is ligated tothe second nucleic acid molecule.

In some embodiments, the 5′ end of the second nucleic acid molecule isligated to the 3′ end of the first nucleic acid molecule.

In other embodiments, the nucleic acid array further comprises a secondnucleic acid molecule, in which the second nucleic acid hybridizes withthe carrier sequence thereby being immobilized to the nucleic acidarray.

In such embodiments, each carrier sequence further comprises a secondimmobilization sequence at its 5′ end, the second immobilizationsequence allows annealing to its complementary nucleotide sequence; and,

the second nucleic acid molecule in the direction from 5′ to 3′comprises a complement of second immobilization sequence and a capturesequence; the complement of second immobilization sequence hybridizeswith the second immobilization sequence of the carrier sequence to forma double strand;

the capture sequence can hybridize with the whole or part of a nucleicacid to be captured, and comprises: (a) an oligonucleotide sequencecapable of capturing mRNA; and/or, (b) a random or degenerateoligonucleotide sequence; or, (c) a specific sequence for a specifictarget nucleic acid; and, the capture sequence has a free 3′ end toenable the second nucleic acid molecule to function as an extensionprimer.

In certain embodiments, each copy of each kind of carrier sequencecomprises a second nucleic acid molecule hybridized therewith.

In some embodiments, the second immobilization sequence allows annealingto its complementary nucleotide sequence and initiating an extensionreaction (for example, it can be used as a binding site of a bridge PCRprimer).

In certain embodiments, the second immobilization sequence is adjacentto the positioning sequence.

In certain embodiments, the second nucleic acid molecule has amodification of 5′ end. In certain embodiments, the modification isphosphorylation or biotin modification.

In certain embodiments, the second nucleic acid molecule is asingle-stranded nucleic acid sequence. In certain embodiments, thesecond nucleic acid molecule is a single-stranded DNA sequence. Incertain embodiments, the second nucleic acid molecule is asingle-stranded RNA sequence.

In some embodiments, the multiple copies of the carrier sequence are anamplification product formed by amplification of the complementarysequence of the carrier sequence as a template, and the amplification isselected from rolling circle amplification (RCA), bridge PCRamplification, multiple strand displacement amplification (MDA) oremulsion PCR amplification.

In certain embodiments, the multiple copies of the carrier sequence area DNB formed by a concatemer of the carrier sequence. In certainembodiments, the multiple copies of the carrier sequence are a DNBformed by rolling circle amplification using the complementary sequenceof the carrier sequence as a template.

In certain embodiments, the multiple copies of the carrier sequence area DNA cluster formed by a clone population of the carrier sequence.

In some embodiments, the multiple copies of the carrier sequence are aDNA cluster formed by bridge PCR amplification using the complementarysequence of the carrier sequence as a template.

In some embodiments, the multiple copies of the carrier sequence are aDNA cluster formed by emulsion PCR amplification of the complementarysequence of the carrier sequence as a template.

In some embodiments, the multiple copies of the carrier sequence are aDNA cluster formed by multiple strand displacement amplification byusing the complementary sequence of the carrier sequence as a template.

In some embodiments, the first nucleic acid molecule further comprises aunique molecular identifier (UMI) sequence, and the UMI sequence islocated at the 5′ end of the complement of first immobilizationsequence; or, the second nucleic acid molecule further comprises a UMIsequence, and the UMI sequence is located at the 5′ end of the capturesequence;

the UMI sequence is a nucleotide sequence composed of at least 1 (forexample, at least 2, at least 3, at least 4, or at least 5; for example,5 to 100, 5 to 50, 5 to 20, such as 10) nucleotide N, and each N isindependently any one of A, C, G and T.

In certain embodiments, the solid support is a chip. In someembodiments, the solid support can be used as a sequencing platform,such as a sequencing chip. In some embodiments, the solid support is ahigh-throughput sequencing chip, such as a high-throughput sequencingchip used in Illumina, MGI, or Thermo Fisher sequencing platform.

In some embodiments, the oligonucleotide sequence capable of capturingmRNA comprises a sequence capable of hybridizing to a poly-A tail of themRNA. In certain embodiments, the oligonucleotide sequence capable ofcapturing mRNA comprises a poly-T oligonucleotide sequence. In certainembodiments, the poly-T oligonucleotide sequence comprises at least 10(e.g., at least 20) deoxythymidine residues.

In some embodiments, the positioning sequence has a length of greaterthan 1 nt, such as greater than 5 nt. In some embodiments, thepositioning sequence has a length of 5 to 50 nt, such as 10 to 50 nt, 10to 30 nt, or 20 to 30 nt. In some embodiments, the lengths of thepositioning sequences contained in different kinds of carrier sequencesmay be the same or different.

In some embodiments, the capture sequence has a length of greater than 1nt. In certain embodiments, the capture sequence has a length of 1 to100 nt, such as 1 to 50 nt, such as 10 to 30 nt.

In some embodiments, the first immobilization sequence has a length ofgreater than 1 nt, such as greater than 10 nt. In some embodiments, thefirst immobilization sequence has a length of 10 to 200 nt. In someembodiments, the first immobilization sequence has a length of 20 to 100nt, such as 20 to 50 nt.

In some embodiments, the second immobilization sequence has a length ofgreater than 1 nt, such as greater than 10 nt. In some embodiments, thesecond immobilization sequence has a length of 10 to 200 nt, forexample, 10 to 100 nt, 10 to 50 nt, 10 to 30 nt, or 10 to 20 nt.

In a fourth aspect, the present invention provides a kit for detectingspatial information of a nucleic acid in a sample, comprising: (i) thenucleic acid array according to the third aspect, wherein the nucleicacid array does not comprise a second nucleic acid molecule; and, (ii) asecond nucleic acid molecule, the second nucleic acid molecule in thedirection from 5′ to 3′ comprises an immobilization region and a capturesequence;

the capture sequence can hybridize with the whole or part of a nucleicacid to be captured, and comprises: (a) an oligonucleotide sequencecapable of capturing mRNA; and/or, (b) a random or degenerateoligonucleotide sequence; or, (c) a specific sequence for a specifictarget nucleic acid; and, the capture sequence has a free 3′ end toenable the second nucleic acid molecule to function as an extensionprimer.

In some embodiments, the kit comprises: (i) a nucleic acid array, whichcomprises a solid support (e.g., a chip) with multiple kinds of carriersequences attached to its surface, in which each kind of carriersequence occupies a different position in the array, said each kind ofcarrier sequence comprises a plurality of copies of the carriersequence, and the carrier sequence in the direction from 5′ to 3′comprises a positioning sequence and a first immobilization sequence,

the positioning sequence has a unique nucleotide sequence correspondingto the position of the kind of carrier sequence on the array;

the first immobilization sequence allows annealing to its complementarynucleotide sequence and initiating an extension reaction;

the nucleic acid array also comprises a first nucleic acid molecule, thefirst nucleic acid molecule in the direction from 5′ to 3′ comprises: acomplement of first immobilization sequence and a complement ofpositioning sequence, and hybridizes with the first immobilizationsequence and the positioning sequence of the carrier sequence to form adouble strand;

and, (ii) the second nucleic acid molecule, the immobilization region ofwhich comprises a double-stranded DNA sequence.

It is easy to understand that a ligase can be used to ligate the secondnucleic acid molecule described in (ii) to the first nucleic acidmolecule contained in the nucleic acid array described in (i).Therefore, in certain embodiments, the kit further comprises a ligase.

In other embodiments, the kit comprises: (i) a nucleic acid array, whichcomprises a solid support (e.g., a chip) with multiple kinds of carriersequences attached to its surface, in which each kind of carriersequence occupies a different position in the array, said each kind ofcarrier sequence comprises a plurality of copies of the carriersequence, and the carrier sequence in the direction from 5′ to 3′comprises: a second immobilization sequence, a positioning sequence anda first immobilization sequence,

the second immobilization sequence allows annealing to its complementarynucleotide sequence;

the positioning sequence has a unique nucleotide sequence correspondingto the position of the kind of carrier sequence on the array;

the first immobilization sequence allows annealing to its complementarynucleotide sequence and initiating an extension reaction;

the nucleic acid array also comprises a first nucleic acid molecule, thefirst nucleic acid molecule in the direction from 5′ to 3′ comprises: acomplement of first immobilization sequence and a complement ofpositioning sequence, and hybridizes with the first immobilizationsequence and the positioning sequence of the carrier sequence to form adouble strand;

and, (ii) the second nucleic acid molecule, the immobilization region ofwhich comprises a complement of second immobilization sequence.

In certain embodiments, the second immobilization sequence is adjacentto the positioning sequence.

It is easy to understand that, under a condition that allows annealing,the second nucleic acid molecule described in (ii) can hybridize with acomplementary region of the carrier sequence contained in the nucleicacid array described in (i), thus the second nucleic acid molecule canbe ligated to the first nucleic acid molecule by using a ligase.Therefore, in certain embodiments, the kit further comprises a ligase.

In another aspect, the present invention also relates to a use of thenucleic acid array according to the third aspect or the kit according tothe fourth aspect for detecting spatial information of a nucleic acid ina sample, or in the manufacture of a detection reagent for detectingspatial information of a nucleic acid in a sample.

In some embodiments, the spatial information of the nucleic acidcomprises the location, distribution and/or expression of the nucleicacid.

In certain embodiments, the sample is a tissue sample, such as a tissuesample comprising cells. In some embodiments, the sample is a tissuesection. In certain embodiments, the tissue section is prepared from afixed tissue, for example, a formalin-fixed paraffin-embedded (FFPE)tissue or deep-frozen tissue.

In a fifth aspect, the present invention also relates to a nucleic acidarray for detecting spatial information of a nucleic acid in a sample,which comprises a solid support (e.g., a chip) with multiple kinds ofcarrier sequences attached to its surface, in which each kind of carriersequence occupies a different position in the array, said each kind ofcarrier sequence comprises a plurality of copies of the carriersequence, and the carrier sequence in the direction from 5′ to 3′comprises: a capture sequence template, a positioning sequence and afirst immobilization sequence, wherein,

the capture sequence template comprises a complementary sequence of acapture sequence, and the capture sequence can hybridize to the whole orpart of a nucleic acid to be captured, including: (a) an oligonucleotidesequence capable of capturing mRNA; and/or, (b) a random or degenerateoligonucleotide sequence; or, (c) a specific sequence for a specifictarget nucleic acid;

the positioning sequence has a unique nucleotide sequence correspondingto the position of the kind of carrier sequence on the array;

the first immobilization sequence allows annealing to its complementarynucleotide sequence and initiating an extension reaction, and the firstimmobilization sequence also comprises a cleavage site, and the cleavagemay be selected from enzymatic cleavage with nicking enzyme, enzymaticcleavage with USER enzyme, photocleavage, chemical cleavage orCRISPR-based cleavage;

the nucleic acid array also comprises a first nucleic acid molecule(also referred to as a capture probe), and the first nucleic acidmolecule in the direction from 5′ to 3′ comprises a binding region, acleavage region, and a carrier sequence complementary region,

the binding region comprises a linker capable of ligating to the surfaceof the solid support;

the cleavage region comprises a cleavage site;

the carrier sequence complementary region comprises a sequence that canbe complementary to the carrier sequence, which in the direction from 5′to 3′ comprises: a complement of first immobilization sequence, acomplement of positioning sequence, and a capture sequence; and, thecapture sequence has a free 3′ end to enable the first nucleic acidmolecule to function as an extension primer;

and, the carrier sequence complementary region of the first nucleic acidmolecule hybridizes with the carrier sequence to form a double strand.

In certain embodiments, the carrier sequence and the first nucleic acidmolecule are single-stranded nucleic acid sequences. In someembodiments, the carrier sequence and the first nucleic acid moleculeare single-stranded DNA sequences.

In certain embodiments, each copy of each kind of carrier sequencecomprises the aforementioned first nucleic acid molecule hybridizedtherewith.

In some embodiments, the linker of the first nucleic acid molecule is alinking group capable of coupling with an activating group (e.g., NH₂),and the surface of the solid support is modified with the activatinggroup (e.g., NH₂). In certain embodiments, the linker comprises —SH,-DBCO, or —NHS. In some embodiments, the linker is

(Azido-dPEG®8-NHS ester) is attached to the surface of the solidsupport.

In some embodiments, the cleavage site contained in the firstimmobilization sequence is a nicking enzyme cleavage site. In someembodiments, the nicking enzyme is selected from USER, BamHI, BmtI andthe like. In certain exemplary embodiments, the cleavage site is shownin SEQ ID NO: 14.

In some embodiments, the cleavage site contained in the cleavage regionof the first nucleic acid molecule is a site where controlled cleavagecan be performed by a chemical, enzymatic, or photochemical method. Incertain embodiments, the cleavage site is an enzyme cleavage site. Insome embodiments, the cleavage site is a USER enzyme cleavage site(UUU).

In some embodiments, the cleavage region of the first nucleic acidmolecule is different from the cleavage site contained in the firstimmobilization sequence of the carrier sequence.

In certain embodiments, the nucleic acid array is prepared by the methoddescribed in the second aspect.

In some embodiments, the multiple copies of the carrier sequence are anamplification product formed by amplification of a complementarysequence of the carrier sequence as a template, and the amplification isselected from rolling circle amplification (RCA), bridge PCRamplification, multiple strand displacement amplification (MDA) oremulsion PCR amplification.

In certain embodiments, the multiple copies of the carrier sequence area DNB formed by a concatemer of the carrier sequence. In certainembodiments, the multiple copies of the carrier sequence are a DNBformed by rolling circle amplification using a complementary sequence ofthe carrier sequence as a template.

In certain embodiments, the multiple copies of the carrier sequence area DNA cluster formed by a clone population of the carrier sequence.

In some embodiments, the multiple copies of the carrier sequence are aDNA cluster formed by bridge PCR amplification using a complementarysequence of the carrier sequence as a template.

In some embodiments, the multiple copies of the carrier sequence are aDNA cluster formed by emulsion PCR amplification of a complementarysequence of the carrier sequence as a template.

In some embodiments, the multiple copies of the carrier sequence are aDNA cluster formed by multiple strand displacement amplification byusing a complementary sequence of the carrier sequence as a template.

In some embodiments, the carrier sequence further comprises a complementof UMI sequence located downstream of the capture sequence template andupstream of the first immobilization sequence, the complement of UMIsequence is complementary to the UMI sequence, and the UMI sequence is anucleotide sequence composed of at least 1 (for example, at least 2, atleast 3, at least 4, or at least 5; for example, 5 to 100, 5 to 50, 5 to20, such as 10) nucleotide N, and each N is independently any one of A,C, G and T;

and, the carrier sequence complementary region of the first nucleic acidmolecule further comprises the UMI sequence located upstream of thecapture sequence and downstream of the complement of firstimmobilization sequence.

In some embodiments, the complement of UMI sequence is located betweenthe positioning sequence and the capture sequence template, or betweenthe first immobilization sequence and the positioning sequence.

In some embodiments, each copy of each kind of carrier sequence (i.e.,the carrier sequences comprising same positioning sequence) has adifferent complement of UMI sequence. Correspondingly, the first nucleicacid molecule (capture probe) hybridized with the carrier sequence ofeach copy also has a different UMI sequence.

In some embodiments, the carrier sequence is removed from the nucleicacid array through the cleavage site contained in the firstimmobilization sequence of the carrier sequence. In such embodiments,the nucleic acid array comprises a solid support (e.g., a chip) withmultiple kinds of capture probes (first nucleic acid molecules) attachedto its surface, and each kind of capture probe (first nucleic acidmolecule) occupies a different position in the array, and is oriented tohave free 3′ end to enable the capture probe (first nucleic acidmolecule) to function as an extension primer, wherein each kind ofcapture probe (first nucleic acid molecule) in the direction from 5′ to3′ comprises: a binding region, an cleavage region, a complement ofpositioning sequence and a capture sequence, wherein,

the binding region comprises a linker capable of ligating to the surfaceof the solid support;

the cleavage region comprises a cleavage site;

the positioning sequence corresponds to the position of the kind ofcapture probe on the array;

the capture sequence can hybridize with the whole or part of a nucleicacid to be captured, and comprises: (1a) an oligonucleotide sequencecapable of capturing mRNA; and/or, (1b) a random or degenerateoligonucleotide sequence; or, (c) a specific sequence for a specifictarget nucleic acid.

In some embodiments, each capture probe of said each kind of captureprobe (i.e., the capture probes comprising the same positioningsequence/complement of positioning sequence) has a different UMIsequence, and the UMI sequence is located upstream of the capturesequence and downstream of the cleavage region. In some embodiments, theUMI sequence is located at the 5′ end of the capture sequence, forexample between the capture sequence and the complement of positioningsequence. In other embodiments, the UMI sequence is located at the 5′end of the complement of positioning sequence, for example, between thecleavage region and the complement of positioning sequence.

In some embodiments, said each kind of carrier sequence (i.e., thecarrier sequences comprising the same positioning sequence) or each kindof capture probe (i.e., the capture probes comprising the samepositioning sequence/complement of positioning sequence) occupies anarea (i.e., active region) having a diameter of less than 1 micrometer,for example, about 900 nanometers, about 800 nanometers, about 700nanometers, about 600 nanometers, or about 500 nanometers, on thesurface of the solid support. In certain embodiments, said each kind ofcarrier sequence or each kind of capture probe has an active region witha diameter of about 500 nanometers.

In certain embodiments, the solid support is a chip. In someembodiments, the solid support can be used as a sequencing platform. Insome embodiments, the solid support is a sequencing chip (MGI), such asBGISEQ-500 platform. In some embodiments, the solid support is ahigh-density array chip, which can be obtained, for example, by themethod described in patent CN103180496B.

In some embodiments, the first immobilization sequence has a length ofgreater than 1 bp, such as greater than 10 bp, or greater than 20 bp. Insome embodiments, the first immobilization sequence has a length of 20to 100 bp, such as 20 to 80 bp.

In some embodiments, the positioning sequence has a length of greaterthan 1 bp, such as greater than 10 bp. In some embodiments, thepositioning sequence has a length of 10 to 100 bp, such as 10 to 50 bp,such as 10 to 30 bp, such as 20 bp.

In some embodiments, the oligonucleotide sequence capable of capturingmRNA comprises a sequence capable of hybridizing to a poly-A tail of themRNA. In certain embodiments, the oligonucleotide sequence capable ofcapturing mRNA comprises a poly-T oligonucleotide sequence. In certainembodiments, the poly-T oligonucleotide sequence comprises at least 10(e.g., at least 20) deoxythymidine residues.

In some embodiments, the capture sequence has a length of greater than 1bp. In some embodiments, the capture sequence is 1 to 100 bp in length,such as 10 to 50 bp, such as 10 to 30 bp.

Detection Method

In a sixth aspect, the present invention provides a method for detectingspatial information of a nucleic acid in a sample, which comprises thefollowing steps:

(1) providing the nucleic acid array according to the third aspect, orobtaining a nucleic acid array by the method according to the firstaspect; wherein,

the nucleic acid array comprises multiple kinds of carrier sequencesattached to a surface of a solid support (e.g., a chip), each kind ofcarrier sequence occupies a different position in the array, and saideach kind of carrier sequence comprises a plurality of copies of thecarrier sequence;

each copy of carrier sequence comprises a first nucleic acid moleculeand a second nucleic acid molecule hybridized therewith, and the firstnucleic acid molecule and the second nucleic acid molecule are notligated to each other;

the first nucleic acid molecule comprises a complement of positioningsequence which is corresponding to the position of the kind of carriersequence on the array,

the second nucleic acid molecule comprises a capture sequence capable ofcapturing the nucleic acid in the sample;

(2) contacting the nucleic acid array with the sample to be tested undera condition that allows annealing, so that the nucleic acid in thesample to be tested anneals to the capture sequence of the secondnucleic acid molecule, and the position of the nucleic acid can becorrelated with the position of the carrier sequence on the nucleic acidarray;

(3) (i) ligating the first nucleic acid molecule and the second nucleicacid molecule that are hybridized to each copy of carrier sequence (forexample, using a ligase);

performing a primer extension reaction by using the ligated first andsecond nucleic acid molecules as a primer, and using the capturednucleic acid molecule as a template under a condition that allows theprimer extension, so as to produce an extension product, in which astrand that hybridizes with the captured nucleic acid molecule has thecomplement of positioning sequence contained in the first nucleic acidmolecule as a spatial information tag; and/or,

performing a primer extension reaction by using the captured nucleicacid molecule as a primer, and using the ligated first and secondnucleic acid molecules as a template under a condition that allows theprimer extension, so as to produce an extended captured nucleic acidmolecule, in which the extended captured nucleic acid molecule has thepositioning sequence as a spatial information tag;

alternatively, (ii) performing a primer extension reaction by using thesecond nucleic acid molecule as a primer and using the captured nucleicacid molecule as a template under a condition that allow the primerextension, so as to produce an extended second nucleic acid molecule, inwhich the extended second nucleic acid molecule comprises acomplementary sequence of the captured nucleic acid; ligating the firstnucleic acid molecule and the extended second nucleic acid molecule thatare hybridized to the each copy of carrier sequence (for example, usinga ligase), such that the extended second nucleic acid molecule which isligated to the first nucleic acid molecule has the complement ofpositioning sequence contained in the first nucleic acid molecule as aspatial information tag;

(4) releasing at least part of the nucleic acid molecules with thespatial information tags from the surface of the array, wherein the partcomprises the positioning sequence or its complementary strand and thecaptured nucleic acid molecule or its complementary strand; and

(5) directly or indirectly analyzing the sequence information of thenucleic acid molecule released in step (4).

In such embodiments, before the target nucleic acid is captured in step(2), the first nucleic acid molecule is not ligated to the secondnucleic acid molecule on the nucleic acid array.

In a seventh aspect, the present invention provides a method fordetecting spatial information of a nucleic acid in a sample, whichcomprises the following steps:

(1) providing the nucleic acid array according to the first aspect, orobtaining a nucleic acid array by the method according to the thirdaspect; wherein,

the nucleic acid array comprises multiple kinds of carrier sequencesattached to a surface of a solid support (e.g., a chip), each kind ofcarrier sequence occupies a different position in the array, and saideach kind of carrier sequence comprises a plurality of copies of thecarrier sequence;

each copy of carrier sequence comprises a first nucleic acid moleculehybridized therewith, and the first nucleic acid molecule is ligated toa second nucleic acid molecule;

the first nucleic acid molecule comprises a complement of positioningsequence which is corresponding to the position of the kind of carriersequence on the array,

the second nucleic acid molecule comprises a capture sequence capable ofcapturing the nucleic acid in the sample;

(2) contacting the nucleic acid array with the sample to be tested undera condition that allows annealing, so that the nucleic acid in thesample to be tested anneals to the capture sequence of the secondnucleic acid molecule, and the position of the nucleic acid can becorrelated with the position of the carrier sequence on the nucleic acidarray;

(3) (iii) perform a primer extension reaction by using the ligated firstand second nucleic acid molecules as a primer, and using the capturednucleic acid molecule as a template under a condition that allows theprimer extension, so as to produce an extension product, in which astrand hybridized with the captured nucleic acid molecule has thecomplement of positioning sequence contained in the first nucleic acidmolecule as a spatial information tag; and/or,

perform a primer extension reaction by using the captured nucleic acidmolecule as a primer, and using the ligated first and second nucleicacid molecules as a template under a condition that allows the primerextension, so as to produce an extended captured nucleic acid molecule,in which the extended captured nucleic acid sequence has the positioningsequence as a spatial information tag;

(4) releasing at least part of the nucleic acid molecules with thespatial information tags from the surface of the array, wherein the partcomprises the positioning sequence or its complementary strand and thecaptured nucleic acid molecule or its complementary strand; and

(5) directly or indirectly analyzing the sequence information of thenucleic acid molecule released in step (4).

In such embodiments, before the target nucleic acid is captured in step(2), the first nucleic acid molecule has been ligated to the secondnucleic acid molecule on the nucleic acid array.

In certain embodiments of the method of the sixth or seventh aspect, themultiple copies of the carrier sequence are a DNB formed by a concatemerof the carrier sequence, or the multiple copies of the carrier sequenceis a DNA cluster formed by a clone population of the carrier sequence.

In certain embodiments of the method of the sixth or seventh aspect, thecarrier sequence and the first nucleic acid molecule are single-strandedDNAs. In certain embodiments, the second nucleic acid molecule issingle-stranded DNA or single-stranded RNA.

In an eighth aspect, the present invention provides a method fordetecting spatial information of a nucleic acid in a sample, whichcomprises the following steps:

(1) providing the nucleic acid array according to the fifth aspect, orobtaining a nucleic acid array by the method according to the secondaspect; wherein the nucleic acid array comprises multiple kinds ofcarrier sequences attached to a surface of a solid support (e.g., achip), each kind of carrier sequence occupies a different position inthe array, and said each kind of carrier sequence comprises multiplecopies of the carrier sequence;

each copy of carrier sequence comprises a first nucleic acid moleculehybridized therewith, and the first nucleic acid molecule comprises acomplement of positioning sequence which is corresponding to theposition of the kind of carrier sequence on the array and a capturesequence capable of capturing the nucleic acid in the sample;

(2) contacting the nucleic acid array with the sample to be tested undera condition that allows annealing, so that the nucleic acid in thesample to be tested anneals to the capture sequence of the first nucleicacid molecule, and the position of the nucleic acid can be correlatedwith the position of the first nucleic acid molecule on the nucleic acidarray;

(3) performing a primer extension reaction by using the first nucleicacid molecule as a primer and using the captured nucleic acid moleculeas a template under a condition that allows the primer extension, so asto produce an extension product, in which a strand hybridized with thecaptured nucleic acid molecule has the complement of positioningsequence contained in the first nucleic acid molecule as a spatialinformation tag;

(4) releasing at least part of the nucleic acid molecules with thespatial information tags from the surface of the array, wherein the partcomprises the positioning sequence or its complementary strand and thecaptured nucleic acid molecule or its complementary strand; and

(5) directly or indirectly analyzing the sequence information of thenucleic acid molecule released in step (4).

In some embodiments, before step (2), the method further comprisesperforming cleavage at the cleavage site contained in the firstimmobilization sequence of the carrier sequence to digest the carriersequence, and at the same time, ligating the first nucleic acid molecule(capture probe) to the surface of the solid support (e.g., a chip). Insuch embodiments, the nucleic acid array comprises multiple kinds ofcapture probes attached to the surface of the solid support (e.g.,chip), each kind of capture probe occupies a different position in thearray, and the capture probe comprises a complement of positioningsequence which is corresponding to the position of the kind of captureprobe on the array and a capture sequence capable of capturing thenucleic acid in the sample;

(2) contacting the nucleic acid array with the sample to be tested undera condition that allows annealing, so that the nucleic acid in thesample to be tested anneals to the capture sequence of the captureprobe, and the position of the nucleic acid can be correlated with theposition of the capture probe on the array;

(3) performing a primer extension reaction by using the capture probe asa primer and using the captured nucleic acid molecule as a templateunder a condition that allows the primer extension, in which theresulting extension product comprises the complement of positioningsequence as a spatial information tag and a complementary sequence ofthe captured nucleic acid molecule, thereby generating a DNA moleculewith spatial information tag; optionally, generating a complementarystrand of the DNA molecule with spatial information tag, and/oroptionally, amplifying the DNA molecule with spatial information tag;

(4) releasing at least part of the DNA molecules with spatialinformation tags and/or their complements or amplicons from the surfaceof the array, wherein the part comprises the spatial information tag orits complementary strand; and

(5) directly or indirectly analyzing the sequence information of thenucleic acid molecule released in step (4).

In some embodiments of the method of the eighth aspect, the firstnucleic acid molecule and the capture probe are DNA molecules, such assingle-stranded DNAs.

In certain embodiments of the method of any one of the sixth to eighthaspects, the spatial information of the nucleic acid comprises thelocation, distribution and/or expression of the nucleic acid.

In certain embodiments of the method of any one of the sixth to eighthaspects, the sample is a tissue sample, such as a tissue section. Incertain embodiments, the tissue section is prepared from a fixed tissue,for example, a formalin-fixed paraffin-embedded (FFPE) tissue ordeep-frozen tissue.

In certain embodiments of the method of any one of the sixth to eighthaspects, the method is used for a non-diagnostic purpose.

In some embodiments of the method described in any one of the sixth toeighth aspects, any nucleic acid analysis method can be used in step(5). In certain embodiments, this step may comprise sequencing. In someembodiments, sequence-specific analysis methods can be used. For examplea sequence-specific amplification reaction may be performed, for exampleusing primers which are specific for the positioning domain and/or for aspecific target sequence (e.g. a particular target DNA to be detected).An exemplary analysis method is a sequence-specific PCR reaction.Therefore, in certain embodiments, this step may comprise asequence-specific PCR reaction.

In some embodiments of the method described in any one of the sixth toeighth aspects, the sequence analysis information obtained in step (5)can be used to obtain spatial information (i.e., location information)of the nucleic acid in the sample. In some embodiments, this spatialinformation may be derived from the nature of the sequence analysisinformation determined, for example it may reveal the presence of aparticular nucleic acid which may itself be spatially informative in thecontext of the tissue sample used, and/or the spatial information (e.g.,spatial localization) may be derived from the position of the tissuesample on the array, coupled with the sequencing information. Therefore,the method may involve simply correlating the sequence analysisinformation to a position in the tissue sample e.g. by virtue of thepositioning tag and its correlation to a position in the tissue sample.In some embodiments, spatial information may conveniently be obtained bycorrelating the sequence analysis data to an image of the tissue sample.Therefore, in such embodiments, the method of any one of the sixth toeighth aspects further comprises step (6): correlating the sequenceanalysis information obtained in step (5) with an image of the sample,wherein the sample is imaged before or after step (3). In someembodiments, the imaging of the sample uses light, bright field, darkfield, phase contrast, fluorescence, reflection, interference, confocalmicroscopy or a combination thereof.

In certain embodiments of the method of the sixth aspect, the method isused to detect a transcriptome in the sample. In such an embodiment, instep (3)(i), a cDNA molecule is generated from the captured RNA moleculeby using the ligated first and second nucleic acid molecules as areverse transcription primer, said cDNA molecule has the complement ofpositioning sequence contained in the first nucleic acid molecule as aspatial information tag, and optionally, the cDNA molecule is amplified;or, in step (3)(ii), a cDNA molecule is generated from the captured RNAmolecule by using the second nucleic acid molecule as a reversetranscription primer, and the first nucleic acid molecule and the cDNAmolecule which are hybridized to each carrier sequence are ligated (forexample, using a ligase) to generate a cDNA molecule having thecomplement of positioning sequence contained in the first nucleic acidmolecule as a spatial information tag, and optionally, the cDNA moleculeis amplified; and, in step (4), at least part of the cDNA moleculesand/or their amplicons are released from the surface of the array,wherein the released nucleic acid molecule may be the first and/orsecond strand of the cDNA molecule or an amplicon thereof, and whereinthe part comprises the spatial information sequence or its complementarystrand. In some embodiments, in step (1), the capture sequence comprisesan oligonucleotide sequence capable of capturing mRNA.

In certain embodiments of the method of the seventh aspect, the methodis used to detect a transcriptome in the sample. In such embodiments, instep (3)(iii), a cDNA molecule is generated from the captured RNAmolecule by using the ligated first and second nucleic acid molecules asa reverse transcription primer, the cDNA molecule has the complement ofpositioning sequence contained in the first nucleic acid molecule as aspatial information tag, and optionally, the cDNA molecule is amplified;and, in step (4), at least part of the cDNA molecules and/or theiramplicons are released from the surface of the array, wherein thereleased nucleic acid molecule may be the first and/or second strand ofthe cDNA molecule or an amplicon thereof, and wherein the part comprisesthe spatial information sequence or a complementary strand thereof. Insome embodiments, in step (1), the capture sequence comprises anoligonucleotide sequence capable of capturing mRNA.

In certain embodiments of the method of the eighth aspect, the method isused to detect a transcriptome in the sample. In such an embodiment, instep (3), a cDNA molecule is generated from the captured RNA molecule byusing the capture probe as an RT primer, the cDNA molecule has a spatialinformation tag, and optionally, the cDNA molecule is amplified; in step(4), at least part of the cDNA molecules and/or their amplicons arereleased from the surface of the array, wherein the released nucleicacid molecule may be the first and/or second strand of the cDNA moleculeor an amplicon thereof, and wherein the part comprises the spatialinformation tag sequence or its complementary strand. In someembodiments, in step (1), the capture sequence comprises anoligonucleotide sequence capable of capturing mRNA.

In some embodiments of the method of any one of the sixth to eighthaspects, before or after the nucleic acid molecule (for example, DNAmolecule) with spatial information tag or the cDNA molecule with spatialinformation tag is released from the surface of the array, thecomplementary strand or the second strand cDNA is generated.

The step for generating the second strand DNA (for example, cDNA) can beperformed in situ on the array, either as a separate step of secondstrand synthesis, or in the initial step of an amplification reaction.Alternatively, the first strand DNA, e.g. cDNA (i.e., the strandgenerated by using the captured nucleic acid molecule as a template) canbe released from the array, and then the second strand synthesis can beperformed, e.g. in a reaction carried out in solution, whether as aseparate step or in an amplification reaction.

When the second strand synthesis is performed on the array (i.e. insitu), the method may comprise an optional step of removing the capturednucleic acid molecule (e.g., RNA) before the second strand synthesis,for example, by using an RNA digesting enzyme (RNase) e.g. RNase H.Procedures for this are well known and described in the art. However,this step is generally unnecessary, and in most cases, RNA will degradenaturally. A step of removing the sample from the array generally alsoremoves RNA from the array.

In some embodiments, the second strand of DNA (e.g., cDNA) is producedin a single reaction, and the second strand synthesis can be performedby any suitable method known in the art. For example, the first strandcDNA which is released from the array substrate, may be incubated withrandom primers, e.g. hexamer primers, and a DNA polymerase, e.g. astrand displacement polymerase, to perform a DNA synthesis reactionusing the first strand as a template. Therefore, in certain embodiments,the synthesis of the complementary strand or the second strand uses arandom primer and a strand displacement polymerase.

In some embodiments of the method according to any one of the sixth toeighth aspects, before the sequence analysis, a step of amplifying thenucleic acid molecules (e.g., DNA molecule) or cDNA molecules with thespatial information tags is further comprised. In some embodiments, theamplification step is performed after the nucleic acid molecules (e.g.,DNA molecules) or cDNA molecules with the spatial information tags arereleased from the array, or the amplification step is performed in situon the array (i.e., when the first nucleic acid molecules and/or carriersequences and/or capture probes are still ligated to the surface of thesolid support). In certain embodiments, the amplification step comprisesPCR.

In some embodiments of the method described in any one of the sixth toeighth aspects, in step (4), the molecule is released from the surfaceof the array by the following method: (i) nucleic acid cleavage; (ii)denaturation; and/or (iii) physical method. In certain embodiments, themolecule is released by applying heated water or a buffer to the solidsupport.

In some embodiments, a step of purifying the released molecule isfurther comprised before sequencing.

In some embodiments, after the sample is contacted with the array andbefore step (3), a step of replenishing the sample with water is furthercomprised.

In some embodiments, before step (4), the method further comprises astep of washing the array to remove residual sample (e.g., tissue).

In certain embodiments, the array comprises at least one orientationmarker to orient the sample on the array.

In some embodiments, in step (5), the sequence analysis step comprises asequencing step. In some embodiments, the sequencing step comprises asequencing reaction based on reversible dye-terminators.

The method for detecting nucleic acid spatial information according toany one of the sixth to eighth aspects of the present invention can beused for RNA detection, transcriptome analysis, DNA detection, genomeanalysis, and the like. Spatial information is of great significance totranscriptomics and genomics related researches, especially useful inthe study of transcriptomic or genomic variation in different cells orregions of tissues, such as comparative study of normal and diseasedcells or tissues, or study of transcriptomic or genomic changes duringdisease process, etc.

For example, the pathophysiological analysis of Alzheimer's diseaseshows that its pathological process involves the interaction of neuronsand glial cells, and the related transcriptome and epigenome studieshave also found that the brain of patient with Alzheimer's disease hasseverely damaged neuronal function and abnormality in innate immuneresponse. However, population-level research cannot reveal thecomplexity of changes between cells and within cell populations,especially for those rare cell types. Ordinary researches at single-celllevel cannot distinguish the characteristics of specific cell types indifferent tissue regions at the same period and the changes in cellcomposition during neurodegeneration. Therefore, in order to furtherreveal the pathogenic mechanism and development mode of diseases, it isurgent to obtain single-cell transcriptome information with spatialdimensions.

The method for detecting nucleic acid spatial information according toany one of the sixth to eighth aspects of the present invention canimmobilize the nucleic acid molecules in different regions of braintissue sample to a chip through the capture sequence with position tagthat is ligated to the chip, and perform sequencing, so thattranscriptome results comprising accurate location information areobtained to realize the detection of changes in specific cell types indifferent regions during the progress of Alzheimer's disease. Inparticular, since the active region of DNB or DNA cluster on the chip ofthe present invention is of a grade as low as nanometers, while the celldiameter is about 12 um, the chip of the present invention can obtainspatial positioning information with subcellular resolution.

The present invention also comprises the following exemplaryembodiments:

Item 1. A method for generating a nucleic acid array, the nucleic acidarray is used to detect spatial information of a biomolecule (e.g., anucleic acid) in a sample, the method comprising the following steps:

(1) providing a circular nucleic acid template, the circular nucleicacid template comprises a template sequence of a kind of capture probe,and the template sequence in the direction from 5′ to 3′ comprises alinker region, a spatial tag region, and a capture region; wherein,

the linker region comprises a cleavage site, and the cleavage may beselected from enzymatic cleavage with nicking enzyme, enzymatic cleavagewith USER enzyme, photocleavage, chemical cleavage or CRISPR-basedcleavage;

the spatial tag region comprises a spatial tag sequence, and the spatialtag sequence corresponds to the position of the kind of capture probe onthe array;

the capture region comprises a capture sequence capable of capturing thebiomolecule (e.g., nucleic acid) in the sample; wherein, the capturesequence comprises: (1a) an oligonucleotide sequence capable ofcapturing a mRNA; and/or, (1b) a random or degenerate oligonucleotidesequence; or, (c) a specific sequence for a specific target molecule(e.g., a target nucleic acid);

(2) performing rolling circle amplification (RCA) by using the circularnucleic acid template as a template to obtain a DNA nanoball (DNB) whichis formed by a concatemer of a complementary sequence of the templatesequence (i.e., template complementary sequence);

(3) ligating the DNB to a surface of a solid support (e.g., a chip);

(4) providing a probe primer, and using the template complementarysequence contained in the DNB as a template to perform a primerextension reaction to produce an extension product, wherein a strandhybridized to the template complementary sequence is a capture probe;optionally, amplifying the extension product; in which the probe primerin the direction from 5′ to 3′ comprises a binding region, an cleavageregion and a primer linker region; wherein,

the binding region comprises a linker that can be ligated to the surfaceof the solid support;

the cleavage region comprises a cleavage site;

the primer linker region is complementary to the whole or part of thesequence of the linker region of the template complementary sequencecontained in the DNB (i.e., the complementary sequence of the linkerregion of the template sequence), and has a free 3′ end to enable theprobe primer to function as a primer and initiate an extension reaction;preferably, the primer linker region comprises a sequence of the linkerregion of the template sequence or a fragment thereof;

(5) ligating the probe primer to the surface of the solid support;wherein, steps (4) and (5) are performed in any order;

(6) performing cleavage at the cleavage site contained in the linkerregion to digest the DNB, so that the extension product in step (4) isseparated from the template DNB that forms the extension product,thereby ligating the capture probe to the surface of the solid support(e.g., chip);

preferably, the circular nucleic acid template, DNB and capture probeare DNA;

preferably, multiple kinds of circular nucleic acid templates areprovided in step (1), and each kind of circular nucleic acid templatecomprises a different template sequence of capture probe, so as toobtain a solid support (e.g., chip) with multiple kinds of captureprobes attached to its surface.

Item 2. The method according to Item 1, wherein the cleavage sitecontained in the linker region is a cleavage site for nicking enzyme;

preferably, the nicking enzyme is selected from USER, BamHI, and BmtI.

Item 3. The method according to item 1 or 2, wherein the linker regionfurther comprises a sequencing primer hybridization region and/or anamplification primer hybridization region; wherein the sequencing primerhybridization region allows annealing to a sequencing primer andinitiating a sequencing reaction, and the amplification primerhybridization region allows annealing to an amplification primer andinitiating an extension and amplification reaction.

Item 4. The method according to any one of items 1 to 3, wherein theoligonucleotide sequence capable of capturing mRNA comprises a sequencecapable of hybridizing with a poly-A tail of the mRNA;

preferably, the oligonucleotide sequence capable of capturing mRNAcomprises a poly-T oligonucleotide sequence;

preferably, the poly-T oligonucleotide sequence comprises at least 10(for example, at least 20) deoxythymidine residues.

Item 5. The method according to any one of items 1 to 4, wherein thetemplate sequence further comprises a probe tag region located upstreamof the capture region and downstream of the linker region, and the probetag region comprises a probe tag complementary sequence which iscomposed of modified bases, and the modified bases are capable ofcomplementary pairing by hydrogen bonds with multiple kinds of mainbases (e.g., C, G, A, T, U);

preferably, the probe tag region is located between the spatial tagregion and the capture region, or between the linker region and thespatial tag region;

preferably, the probe tag complementary sequence comprises a pluralityof (for example, at least 10) Inosines.

Item 6. The method according to any one of items 1 to 5, which has oneor more of the following characteristics:

(i) the linker region has a length of greater than 1 bp, for example,greater than 10 bp, or greater than 20 bp; preferably, the linker regionhas a length of 20 to 100 bp;

(ii) the spatial tag region has a length of greater than 1 bp, forexample, greater than 10 bp; preferably, the spatial tag region has alength of 10 to 100 bp;

(iii) the capture region has a length of greater than 1 bp; preferably,the capture region has a length of 1-100 bp;

(iv) the probe tag region has a length of greater than 1 bp, forexample, greater than 5 bp; preferably, the probe tag region has alength of 5-100 bp.

Item 7. The method according to any one of items 1 to 6, wherein thesolid support is a chip;

preferably, the solid support can be used as a sequencing platform, suchas a sequencing chip.

Item 8. The method according to any one of items 1 to 7, wherein, instep (4), the complementary sequence of the spatial tag sequence issequenced while the primer extension reaction is performed, so as toobtain the sequence information of the spatial tag sequence contained inthe corresponding capture probe.

Item 9. The method according to any one of items 1 to 7, wherein, beforestep (4), a step of sequencing the complementary sequence of the spatialtag sequence contained in the DNB is further comprised;

preferably, after the sequencing is completed, dNTP added to thesynthetic strand due to the sequencing is removed by washing.

Item 10. The method according to any one of items 1 to 9, wherein thelinker is a linking group capable of coupling with an activated group(e.g., NH₂), and the solid support is modified by the activated group(e.g., NH₂) on its surface;

preferably, the linker comprises —SH, -DBCO or —NHS;

preferably, the linker is

(Azido-dPEG®8-NHS ester) is attached to the surface of the solidsupport.

Item 11. The method according to any one of items 1 to 10, wherein thecleavage site contained in the cleavage region is a site wherecontrolled cleavage can be performed by a chemical, enzymatic orphotochemical method;

preferably, the cleavage site is an enzyme cleavage site;

preferably, the cleavage sites contained in the cleavage region and thelinker region are different.

Item 12. The method according to any one of items 1 to 11, wherein theamplification comprises PCR.

Item 13. A nucleic acid array prepared by the method according to anyone of items 1 to 12.

Item 14. A nucleic acid array for detecting spatial information of abiomolecule (e.g., a nucleic acid) in a sample, which comprises a solidsupport (e.g., a chip) with multiple kinds of capture probes attached toits surface, in which each kind of capture probe occupies a differentposition in the array and is oriented to have free 3′ end to enable thecapture probe to function as an extension primer, wherein each kind ofcapture probe in the direction from 5′ to 3′ comprises: a bindingregion, an cleavage region, a spatial tag sequence and a capturesequence, wherein,

the binding region comprises a linker that can be ligated to the surfaceof the solid support;

the cleavage region comprises a cleavage site;

the spatial tag sequence corresponds to the position of the kind ofcapture probe on the array;

the capture sequence is capable of hybridizing with the whole or part ofthe biomolecule (e.g., nucleic acid) to be captured, and comprises: (1a)an oligonucleotide sequence capable of capturing mRNA; and/or, (1b) arandom or degenerate oligonucleotide sequence; or, (c) a specificsequence for a specific target molecule (e.g., a target nucleic acid).

Item 15. The nucleic acid array according to Item 14, wherein eachcapture probe of the each kind of capture probe (i.e., capture probescomprising the same spatial tag sequence) has a different probe tagsequence, and the probe tag sequence is located upstream of the capturesequence and downstream of the cleavage region;

preferably, the probe tag sequence is located between the capturesequence and the spatial tag sequence, or between the cleavage regionand the spatial tag sequence.

Item 16. The nucleic acid array according to Item 14 or 15, wherein theeach kind of capture probe (i.e., capture probes comprising the samespatial tag sequence) occupies an area (i.e., active region) with adiameter of less than 1 micron on the surface of the solid support;

preferably, the each kind of capture probe occupies an active regionwith a diameter of about 500 nanometers.

Item 17. The nucleic acid array according to any one of items 14 to 16,wherein the solid support is a chip;

preferably, the solid support can be used as a sequencing platform, suchas a sequencing chip.

Item 18. The nucleic acid array according to any one of items 14 to 17,wherein the nucleic acid array is prepared by the method according toany one of items 1 to 12.

Item 19. A method for detecting spatial information of a biomolecule ina sample, which comprises the following steps:

(1) providing the nucleic acid array according to any one of items 13 to18, or obtaining a nucleic acid array by the method according to any oneof items 1 to 12; the nucleic acid array comprises multiple kinds ofcapture probes attached to a surface of a solid support (e.g., a chip),each kind of capture probe occupies a different position in the array,and the capture probe comprises a spatial tag sequence corresponding tothe position of the kind of capture probe on the array and a capturesequence capable of capturing a biomolecule in a sample;

(2) contacting the nucleic acid array with the sample to be tested, sothat the capture sequence of the capture probe binds to the biomoleculein the sample to be tested, and thus the position of the biomolecule canbe correlated with the position of the capture probe on the nucleic acidarray, and a biomolecule labeled by spatial tag is generated;

(3) releasing the biomolecule labeled by spatial tag from the surface ofthe array; and

(4) directly or indirectly analyzing the sequence of the biomoleculereleased in step (3).

Item 20. A method for detecting spatial information of a nucleic acid ina sample, which comprises the following steps:

(1) providing the nucleic acid array according to any one of items 13 to18, or obtaining a nucleic acid array by the method according to any oneof items 1 to 12; the nucleic acid array comprises multiple kinds ofcapture probes attached to a surface of a solid support (e.g., a chip),each kind of capture probe occupies a different position in the array,and the capture probe comprises a spatial tag sequence corresponding tothe position of the kind of capture probe on the array and a capturesequence capable of capturing the nucleic acid in the sample;

(2) contacting the nucleic acid array with the sample to be tested undera condition that allows annealing, so that the nucleic acid in thesample to be tested anneal to the capture sequence of the capture probe,and thus the position of the nucleic acid can be correlated with theposition of the capture probe on the array;

(3) using the capture probe as a primer and using the captured nucleicacid molecule as a template to perform a primer extension reaction undera condition that allows the primer extension, the resulting extensionproduct comprises the spatial tag sequence and a complementary sequenceof the captured nucleic acid molecule, thereby generating a DNA moleculelabeled with spatial tag; optionally, generating a complementary strandof the DNA molecule labeled with spatial tag, and/or optionally,amplifying the DNA molecule labeled with spatial tag;

(4) releasing at least part of the DNA molecules labeled with spatialtags and/or their complementary strands or amplicons from the surface ofthe array, wherein the part comprises the spatial tag sequence or itscomplementary strand; and

(5) directly or indirectly analyzing the sequence of the nucleic acidmolecule released in step (4);

preferably, the spatial information of the nucleic acid comprises thelocation, distribution and/or expression of the nucleic acid;

preferably, the capture probe is a DNA molecule;

preferably, the sample is a tissue sample, such as a tissue section;

preferably, the tissue section is prepared from a fixed tissue, forexample, a formalin-fixed paraffin-embedded (FFPE) tissue or deep-frozentissue.

Item 21. The method according to Item 20, wherein in step (5), thesequence analysis comprises a sequencing or sequence-specific PCRreaction.

Item 22. The method according to Item 20 or 21, wherein the methodfurther comprises step

(6): correlating the sequence analysis information obtained in step (5)with an image of the sample, wherein the sample is imaged before orafter step (3).

Item 23. The method according to any one of items 20 to 22, wherein themethod is used for detecting a transcriptome in the sample, wherein:

in step (3), using the capture probe as a RT primer to synthesize a cDNAmolecule from the captured RNA molecule, in which the cDNA molecule islabeled with a spatial tag, and optionally, the cDNA molecule isamplified;

in step (4), at least part of the cDNA molecules and/or their ampliconsis released from the surface of the array, wherein the released nucleicacid molecule may be a first and/or second strand of the cDNA moleculeor an amplicon thereof, and wherein the part comprises a spatial tagsequence or its complementary strand;

preferably, in step (1), the capture sequence comprises anoligonucleotide sequence capable of capturing mRNA.

Item 24. The method according to any one of items 20 to 23, whereinbefore or after the DNA molecule labeled with spatial tag or the cDNAmolecule labeled with spatial tag is released from the surface of thearray, the complementary strand or the cDNA second strand is generated;

preferably, the synthesis of the complementary strand or second stranduses a random primer and a strand displacement polymerase.

Item 25. The method according to any one of items 20 to 24, wherein,before the sequence analysis, it further comprises a step of amplifyingthe DNA molecule or cDNA molecule that is labeled with spatial tag;

preferably, the amplification step is performed after the DNA or cDNAmolecule labeled with spatial tag is released from the array, or theamplification step is performed in situ on the array;

preferably, the amplification step comprises PCR.

Item 26. The method according to any one of items 20 to 25, wherein thesequence analysis further comprises a step of purifying the releasedmolecule.

Item 27. The method according to any one of items 20 to 26, before step(4), the method further comprises a step of washing the array to removea residue of the sample (for example, tissue).

Item 28. The method according to any one of items 20 to 27, in step (4),the molecule is released from the surface of the array by the followingmethod: (i) nucleic acid cleavage; (ii) denaturation; and/or (iii)physical method;

preferably, the molecule is released from the cleavage region of thecapture probe by enzyme cleavage.

Item 29. The method according to any one of items 20 to 28, in step (6),the sample is imaged by using light, bright field, dark field, phasecontrast, fluorescence, reflection, interference, confocal microscopy ora combination thereof.

BENEFICIAL EFFECT

The present invention provides a novel array for detecting spatialinformation of nucleic acid and a preparation method thereof. When thenucleic acid array is applied to the detection of spatial information ofnucleic acid, high-precision subcellular positioning and high-throughputtissue positioning can be realized at the same time. The array of thepresent invention and the detection method based on the array have greatapplication value in cell positioning, subcellular positioning,organelle positioning, cell interaction, organelle interaction,molecular pathway research, disease diagnosis and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of cDNA synthesis after capturing mRNAin Example 2.

FIG. 2 shows a schematic diagram of the molecule released from the chipin Example 2.

FIG. 3 shows the results of the 2100 detection of cDNA fragmentdistribution in Example 2.

FIG. 4 shows the matching result of the 25 bp sequence of first strandobtained by cDNA sequencing in Example 3 and the fq of the positioningsequence on the capture chip.

FIG. 5 shows a graph of the expression of mRNA in the tissue section inExample 3.

FIG. 6 shows a schematic diagram of the probe primer and the carriersequence contained in the DNB of an exemplary embodiment in Example 4.

FIG. 7 shows a schematic diagram of the probe ligated to the chip inExample 4.

FIG. 8 shows a schematic diagram of cDNA synthesis of the capturednucleic acid molecule in Example 5.

FIG. 9 shows a schematic diagram of the molecule released from the chipin Example 5.

FIG. 10 shows the results of the 2100 detection of cDNA fragmentdistribution in Example 5.

FIG. 11 shows the matching result of the 20 bp sequence of first strandobtained by cDNA sequencing in Example 6 and the fq of the positioningsequence on the capture chip.

FIG. 12 shows a graph of the expression of mRNA in the tissue section inExample 6.

EXAMPLES

The present invention is now described with reference to the followingexamples which are intended to illustrate the invention rather thanlimit the invention.

Unless otherwise specified, the experiments and methods described in theexamples were basically performed according to conventional methods wellknown in the art and described in various references. In addition, forthose without specific conditions in the examples, they were carried outin accordance with the conventional conditions or the conditionsrecommended by the manufacturer. The reagents or instruments usedwithout the manufacturer's indication were all conventional productsthat were purchased commercially. Those skilled in the art know that theexamples describe the present invention by way of example, and are notintended to limit the scope of protection claimed by the presentinvention. All publications and other references mentioned in herein areincorporated by reference in their entirety.

Example 1. Preparation of Capture Chip (1)

1. The following DNA library sequence was designed and synthesized. Thesequence synthesis was performed by Beijing Liuhe BGI.

5′-phosphorylated-AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA (Linker A, SEQ IDNO: 1) NNNNNNNNNNNNNNNNNNNNNNNNNNN (complement of positioning sequence,N represented any base, such as C, G, A or T) CTGATAAGGTCGCCA(complement of second immobilization sequence, SEQ ID NO: 2)CAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT (Linker B, SEQ ID NO: 3)-3′.Wherein, Linker A comprised a part of the complement of firstimmobilization sequence and a circularization site, and Linker Bcomprised another part of the complement of first immobilizationsequence, a cleavage site, and a circularization site.

2. In situ amplification of library

Preparation of DNA nanoball (DNB): 40 ul of the following reactionsystem was prepared, and 80 fmol of the above DNA library was added, inwhich the DNB primer has a sequence of GGCCTCCGACTTAAGTCGGATCGT (SEQ IDNO: 4) and synthesized by Beijing Liuhe BGI.

Final Ingredient Volume (ul) concentration DNA library sequence 80 fmol(X) 10× phi29 buffer (produced by BGI) 4 1× DNB primer sequence, 10 uM 41 uM H₂O 32-x

The above reaction system was placed in a PCR machine for reaction. Thereaction conditions were as follows: 95° C. for 3 min, 40° C. for 3 min;after the reaction, it was placed on ice, added with 40 ul of mixedenzyme I and 2 ul of mixed enzyme II required to prepare DNB in DNBSEQsequencing kit, as well as 1 ul of ATP (100 mM mother liquor, ThermoFisher), and 0.1 ul of T4 ligase (produced by BGI). After mixing well,the above reaction system was transferred to a PCR machine at 30° C. andreacted for 20 minutes to form DNB. The DNB was loaded on BGISEQ500sequencing chip according to the method described in the BGISEQ500 SE50kit.

3. Sequencing and decoding of the positioning sequence: According to theinstructions of the BGISEQ500 SE50 sequencing kit, the positioningsequence is decoded and sequenced, with a sequencing length of 25 bp.The fq file formed by sequencing was stored for later use.

4. Immobilizing capture sequence: the following DNA sequence wassynthesized by Beijing Liuhe BGI: 5′-phosphorylated-CTGATAAGGTCGCCA(complement of second immobilization sequence, SEQ ID NO: 5)NNNNNNNNNN(UMI)TTTTTTTTTTTTTTTTTTTVN (capture sequence, SEQ ID NO:6)-3′, wherein N represented any base (for example, C, G, A, or T). Thesequencing chip was taken from the sequencer, the cleavage reagent ofHole 7 of the BGISEQ500 SE50 kit was pumped into the chip (it wasensured that the reagent covered the entire chip and no bubbles weregenerated). The chip was allowed to stand at 60° C., and reaction wasperformed for 10 minutes. After the reaction, an appropriate amount of5×SSC (purchased from Shanghai Shenggong) was pumped into the sequencingchip to replace the previous reagent in the chip. The capture sequencewas diluted with 5×SSC to 1 uM, and an appropriate amount of the dilutedcapture sequence was added to the chip, so that the chip was filled withthe capture sequence. The chip was allowed to stand for about 30 minutesat room temperature so that the capture sequence fully hybridized withthe DNB.

5. Chip dicing: The prepared chip was cut into several small slices, inwhich the size of the slices was adjusted according to the needs of theexperiment, and the chip was immersed in 50 mM tris buffer with pH8.0,and stored at 4° C. for later use.

Example 2. Capture of Tissue mRNA and cDNA Synthesis

1. Frozen tissue section. The cerebellar tissue sections of mice weremade according to the standard procedure of frozen section.

2. mRNA capture. According to the size of the tissue section, the chipwith suitable size prepared in Example 1 was taken and placed at roomtemperature. After the liquid on the chip was evaporated, the tissuesection was attached to the capture chip by virtue of the temperaturedifference between the tissue section and the chip in the tissuechopper. The attached tissue section was placed at room temperature,5×SSC reaction solution was added to the chip (and fully covered theregion to which the tissue attached), and reaction was performed at 30°C. for 30 minutes to allow the mRNA in the tissue to fully hybridizewith the capture region on the chip.

3. cDNA synthesis. 5×SSC was used to wash the chip twice at roomtemperature, 200 ul of the following reverse transcriptase reactionsystem was prepared, the reaction solution was added to the chip tofully cover it, reaction was performed at 42° C. for 90 min to 180 min.The mRNA would use polyT as primer to perform cDNA synthesis, the 3′ endof mRNA carried TSO tag (AAGTCGGAGGCCAAGCGGTC/rG//rG//iXNA_G/) (SEQ IDNO: 7) for the synthesis of cDNA complementary strand. The structurediagram of the above process was shown in FIG. 1 .

Final Ingredient Volume (ul) concentration Superscript II First strandbuffer 40 1× (5×), Thermo Fisher Betaine (5M), Aladdin 40 1M dNTP (10mM), Thermo Fisher 20 1 mM MgCl₂ (100 mM), Aladdin 15 7.5 mM TSOsequence (50 uM), synthesized 10 1 uM by Beijing Liuhe BGI SuperscriptII RT (200 U/ul), 10 10 U/ul Thermo Fisher DTT(100 mM) 10 5 mM RNaseinhibitor (40 U/ul), 5 1 u/ul Thermo Fisher Nucleic acid-free molecularwater 50 (NF H₂O)

4. Ligating spatial positioning region to capture region. After cDNAsynthesis, the chip was washed twice with 5×SSC. 1 ml of the followingreaction system was prepared, an appropriate volume thereof was pumpedinto the chip to ensure that the chip was filled with the followingligation reaction solution, and the nick shown in FIG. 1 was ligated.Reaction was performed at room temperature for 30 minutes. After thereaction, the chip was washed with 5×SSC at a temperature of 55° C. for3 times, 5 min for each time.

Ingredient Volume (ul) Concentration 10× T4 ligase buffer (produced byBGI) 100 1× T4 ligase (600 U/ul, produced by BGI) 100 60 u/ul Glycerin(Aladdin) 10 10% H₂O 790

5. cDNA release. After first strand of cDNA was synthesized on the chip,an appropriate amount of formamide solution was added to the chip andreacted at 55° C. for 10 minutes to release the cDNA strand from thechip. The released molecule had the structure shown in FIG. 2 . Thereaction solution released from the chip was collected, 2×XP magneticbeads were used to purify the cDNA strand, and finally 45 ul of TEbuffer (Thermo Fisher) was used to recover the product. The qubit ssDNAdetection kit was used to quantitatively detect single-stranded cDNA.

6. cDNA amplification. 100 ul of the following reaction system wasprepared:

Ingredient Volume (ul) Concentrationrecovery product of cDNA first strand 42Rolling circle amplification primer  8 0.8 uM AAGTCGGAGGCCAAGCGGTC (with5′-phosphorylation, SEQ ID NO: 8, 10 uM) (Beijing Liuhe BGI)2x HiFi (produced by BGI) 50 1x

The above reaction system was placed in the PCR machine, and thefollowing reaction program was set: 95° C. for 3 min, 11 cycles (98° C.for 20 s, 58° C. for 20 s, 72° C. for 3 min), 72° C. for 5 min, 4° C.for ∞. After the reaction was completed, XP beads were used to purifyand recover. The qubit kit was used to quantify the concentration ofdsDNA, and the 2100 was used to detect the distribution of cDNAfragments. The 2100 detection results were shown in FIG. 3 . The cDNAlength was normal.

Example 3. Construction and Sequencing of cDNA Library

1. Tn5 interruption. According to the cDNA concentration, 20 ng of cDNAwas added with 0.5 uM of Tn5 enzyme and corresponding buffer (thecoating method for Tn5 enzyme was performed according to stLFR libraryconstruction kit), and mixed well to form 20 ul of reaction system. Thereaction was performed at 55° C. for 10 min, 5 ul of 0.1% SDS was addedand mixed well at room temperature for 5 minutes to end the Tn5interruption step.

2. PCR amplification. 100 ul of the following reaction system wasprepared:

Ingredient Volume (ul) Concentration Product after Tn5 interruption 252x Hifi ready mix (produced by BGI) 50 0.8 uMPrimer AAGTCGGAGGCCAAGCGGTC  4 0.4 uM(5-phosphorylation modification, SEQ IDNO: 9, 10 uM) (Beijing Liuhe BGI)Primer GAGACGTTCTCGACTCAGAAGATG (SEQ ID  4 0.4 uMNO: 10) (synthesized by Beijing Liuhe BGI) NF H₂O 17

After mixing, it was placed in PCR machine, the following program wasset: 95° C. 3 min, 11 cycles (98° C. for 20 s, 58° C. for 20 s, 72° C.for 3 min), 72° C. for 5 min, 4° C. for ∞. After the reaction wascompleted, XP beads were used to purify and recover. The qubit kit wasused to quantify dsDNA concentration.

3. Sequencing. 80 fmol of the amplified product after the aboveinterruption was taken to prepare DNB. 40 ul of the following reactionsystem was prepared:

Ingredient Volume (ul) Final concentrationAmplification product after the above 80 fmol (X) interruption10x phi29 buffer (produced by BGI)  4 1X DNB primer sequence 10 uM  41 uM (GGCCTCCGACTTGAGACGTTCTCG, SEQ ID NO: 11)(synthesized by Beijing Liuhe BGI) H₂O 32-x

The above reaction system was placed in the PCR machine for reaction,and the reaction conditions were as follows: 95° C. for 3 min, 40° C.for 3 min. After the reaction was completed, it was placed on ice, addedwith 40 ul of mixed enzyme I and 2 ul of mixed enzyme II required toprepare DNB in DNBSEQ sequencing kit, as well as 1 ul of ATP (100 mMmother liquor, Thermo Fisher), 0.1 ul of T4 ligase (produced by BGI).After mixing well, the above reaction system was transferred to PCRmachine at 30° C. and reacted for 20 minutes to form DNB. The DNB wasloaded on the sequencing chip of MGISEQ2000 according to the methoddescribed in the PESO kit of MGISEQ2000, and the sequencing wasperformed according to the relevant instructions with the PESOsequencing model, wherein the sequencing of first strand was dividedinto two stages, i.e., sequencing 25 bp and then performing 15 cycles ofdark reaction, then sequencing 10 bp UMI sequence, and 50 bp wassequenced for second strand.

Data Analysis

1. The 25 bp sequence of first strand obtained by cDNA sequencing wasmatched with the fq of the positioning sequence on the capture chip (thesequencing result obtained in step 3 in Example 1) by alignment. Thematching result was shown in FIG. 4 , in which the bright arearepresented the region where the 25 bp of cDNA sequencing exactlymatched the capture chip, and this region represented the region on thecapture chip for tissue capture. It showed that the capture chip coulduse the spatial positioning region to accurately locate the tissuecapture region.

2. The DNB matched to the capture chip by the cDNA sequencing wasfurther analyzed, and the alignment analysis between the second strandsequencing result of cDNA (mRNA expression in reaction tissue) of theseDNB reads and mouse genome was performed. For the DNB aligned to mousegenome, the mouse mRNA information was aligned to the capture chipthrough the 25 bp sequencing result. As shown in FIG. 5 , the left sideshowed the full overall picture of the mRNA expression in the analyzedtissue section, the overall picture showed that this capture chip couldanalyze the mRNA expression differences in tissues; the right side ofthis figure showed the tissue expression level of a randomly selectedgene expressed in mouse cerebellum, which indicated that this chip couldanalyze the expression differences of a certain gene in the wholetissue.

Example 4. Preparation of Capture Chip (2)

1. The following DNA library sequence was designed and synthesized. Thesequence synthesis was performed by Beijing Liuhe BGI.

5′-phosphorylated-GAACGACATGGCTTTTTCCCGTAGCCATGTCGTTCTGCGCCTTC CCGATG(immobilization sequence 1, SEQ ID NO: 12) NNNNNNNNNNNNNNNNNNNNNN(positioning sequence template, N represented any base, for example, C,G, A or T) IIIIIIIIII (UMI template, I represented Inosine)TTTTTTTTTTTTTTTTTTTTT (capture sequence, SEQ ID NO: 13) CCTCAGC(cleavage site, SEQ ID NO: 14) CCTTGGCTCACA (immobilization sequence 2,SEQ ID NO: 15). Wherein, the immobilization sequence 1 comprised apartial sequence of the complement of first immobilization sequence anda circularization site, and the immobilization sequence 2 comprised apartial sequence of the complement of first immobilization sequence anda circularization site.

2. In Situ Amplification of Library

Preparation of DNA nanoball (DNB): 40 ul of the following reactionsystem was prepared, 80 fmol of the above-mentioned DNA library wasadded, the DNB primer has a sequence of GACATGGCTACGTGTGAGCCAAGG (SEQ IDNO: 16), which was synthesized by Beijing Liuhe BGI.

Final Ingredient Volume (ul) concentration DNA library sequence 80 fmol(x) 10× phi29 buffer (produced by BGI) 4 1× DNB primer sequence, 10 uM 41 uM H₂O 32-x

The above reaction system was placed in a PCR machine for reaction, andthe reaction conditions were as follows: 95° C. for 3 min, 40° C. for 3min; after the reaction, it was placed on ice, added with 40 ul of mixedenzyme I and 2 ul of mixed enzyme II required to prepare DNB in DNBSEQsequencing kit, and 1 ul of ATP (100 mM mother liquor, Thermo Fisher),0.1 ul of T4 ligase (produced by BGI). After mixing well, the abovereaction system was transferred to a PCR machine at 30° C. and reactedfor 20 minutes to form DNB. The DNB was loaded on the BGISEQ500sequencing chip according to the method described in the BGISEQ500 SE50kit.

3. Decoding of spatial information

(1) Surface modification of chip:

The surface of the above BGISEQ-500 platform chip was allowed to contactwith Azido-dPEG®8-NHS ester that had a structure as follows:

The chip surface modification was carried out according to the followingmethod: NHS-PEG8-Azido (564.58 g/mol) concentration was 45 μM, and 100ml was prepared by the method:

Reagent Dosage Unit NHS-PEG8-Azido 2.54 mg 1× PBS (pH 7.4) 100 ml

Stored at −20° C., avoided repeated freezing and thawing.

DBCO-primer had a concentration of 1 uM, and diluted with PBS.

(2) Coupling of primer probe:

The following primer probe sequences were synthesized by Beijing LiuheBGI:

DBCO (linking group)-UUU (USER cleavage site) TTTTTCCCGTAGCCATGTCGTTCTGCGCCTTCCCGATG (SEQ ID NO: 17, this sequence comprised a complement offirst immobilization sequence, a PCR amplification site sequence, anintermediate sequence). 1 uM of the above primer probe was diluted withPBS and introduced to the chip modified with azido, and reacted at roomtemperature for 1 hour or overnight.

(3) Decoding of spatial information. According to the instructions ofthe BGISEQ500 SE50 sequencing kit, the spatial information sequence wasdecoded and sequenced with a sequencing length of 30 bp (the first 20 bpwas spatial information sequence, and the last 10 bp was probe tagsequence). The fq file formed by sequencing was stored for later use.

(4) Synthesis of capture region:

A mixed solution of dTTP and Hifi polymerase was prepared, DNB was usedas a template, a probe sequence comprising a spatial positioning regionwas used as a primer, and dTTP was used as a substrate, to extend anoligo dT sequence.

4. Release of probe comprising spatial information

1 uM of Spatial_RNA_BbvCI primer (diluted with 5×SSC) was prepared, theprimer sequence CCTCAGCCAACTCCT (SEQ ID NO: 18) was synthesized byBeijing Liuhe BGI. hybridization was performed at room temperature for30 minutes. BbvCI excision system (1.5 ml) was prepared: 15 ul RE+150 ul10×CS Buffer+1335 ul ddH₂O, and introduced to the chip after the spatialpositioning region was decoded, reaction was performed at 37° C. for 1 hor overnight. Washing was performed twice by adding WB2 of thesequencing kit (MGI), then reaction was performed using formamide at 55°C. for 15 min, followed by washing with WB2 twice. The schematic diagramof the obtained probe was shown in FIG. 7 , and the probe sequence wasas follows:

UUU (cleavage region) TTTTTCCCGTAGCCATGTCGTTCTGCGCCTTCCCGATG (complementof first immobilization sequence, SEQ ID NO: 19) NNNNNNNNNNNNNNNNNNN(complement of positioning sequence, which was the same as thepositioning sequence template in the DNA library sequence in step 1)NNNNNNNNNN (UMI sequence, which was a complementary sequence of therandom base sequence obtained from the UMI template which is used as atemplate in step 1) TTTTTTTTTTTTTTTTTTTTT (capture sequence, SEQ ID NO:20).

5. Chip dicing

The prepared capture chip was cut into several small slices, the size ofthe slices was adjusted according to the needs of the experiment, andthe chip was immersed in 50 mM tris buffer, pH8.0, and stored at 4° C.for later use.

Example 5. Capture of Tissue mRNA and Synthesis of cDNA

1. Frozen tissue section. Cerebellar tissue sections of mice were madeaccording to the standard procedure of frozen section.

2. Capture of mRNA. According to the size of the tissue section, thechip with suitable size prepared in Example 4 was taken and placed atroom temperature. After the liquid on the chip had evaporated, thetissue section was attached to the capture chip by virtue of thetemperature difference between the tissue section and the chip in thetissue chopper. The attached tissue section was placed at roomtemperature, 5×SSC reaction solution was added to the chip (and fullycovered the tissue-attached area), and reaction was performed at 30° C.for 30 minutes to allow the mRNA in the tissue to fully hybridize withthe capture region on the chip.

3. Synthesis of cDNA. 5×SSC was used to wash the chip twice at roomtemperature, 200 ul of the following reverse transcriptase reactionsystem was prepared, the reaction solution was added to the chip tofully cover it, reaction was performed at 42° C. for 90 min to 180 min.mRNA would use polyT as primer for cDNA synthesis, and the 3′ end ofmRNA carried TSO tag (CGTAGCCATGTCGTTCTGCG/rG//rG//iXNA_G/) (SEQ ID NO:21) for the synthesis of cDNA complementary strand. The structurediagram of the above process was shown in FIG. 8 .

Final Ingredient Volume (ul) concentration Superscript II First strandbuffer 40 1× (5×), Thermo Fisher Betaine (5M), Aladdin 40 1M dNTP (10mM), Thermo Fisher 20 1 mM MgCl₂ (100 mM), Aladdin 15 7.5 mM TSOsequence (50 uM), synthesized 10 1 uM by Beijing Liuhe BGI SuperscriptII RT (200 U/ul), 10 10 U/ul Thermo Fisher DTT (100 mM) 10 5 mM RNaseinhibitor (40 U/ul), 5 1 u/ul Thermo Fisher NF H₂O 50

4. Release of cDNA. After the cDNA first strand was synthesized on thechip, a USER enzyme reaction system was prepared, and the reaction wascarried out according to the USER enzyme instruction manual. Thereleased molecule had the structure shown in FIG. 9 . The reactionsolution released from the chip was collected, 2×XP magnetic beads wereused to purify the cDNA first strand, and finally 45 ul of TE buffer(Thermo fisher) was used to recover the product.

5. Amplification of cDNA. 100 ul of the following reaction system wasprepared:

Ingredient Volume (ul) ConcentrationRecovery product of cDNA first strand 42Primer CGTAGCCATGTCGTTCTGCG (with  8 0.8 uM5′-phosphorylation, 10 uM, SEQ ID NO: 22) (Beijing Liuhe BGI)2x HiFi (produced by BGI) 50 1X

The above reaction system was transferred to PCR machine, and thefollowing reaction program was set: 95° C. for 3 min, 11 cycles (98° C.for 20 s, 58° C. for 20 s, 72° C. for 3 min), 72° C. for 5 min, 4° C.for co. After the reaction was completed, XP beads were used to purifyand recover. The qubit kit was used to quantify the concentration ofdsDNA, and the 2100 was used to detect the distribution of cDNAfragments. The 2100 test results were shown in FIG. 10 , in which thecDNA length was normal.

Example 6. Construction and Sequencing of cDNA Library

1. Tn5 interruption. According to the cDNA concentration, 20 ng of cDNAwas added with 0.5 uM of Tn5 enzyme and corresponding buffer (thecoating method for Tn5 enzyme was performed according to the stLFRlibrary construction kit), and mixed well to form 20 ul of reactionsystem. The reaction was performed at 55° C. for 10 min, and 5 ul of0.1% SDS was added and mixed at room temperature for 5 minutes to endthe Tn5 interruption step.

2. PCR amplification. 100 ul of the following reaction system wasprepared:

Ingredient Volume (ul) Concentration Product after Tn5 interruption 252x Hifi ready mix (produced by BGI) 50 0.8 uMPrimer CGTAGCCATGTCGTTCTGCG (with  4 0.4 uM5′-phosphorylation, 10 uM, SEQ ID NO: 23) (Beijing Liuhe BGI)Primer GAGACGTTCTCGACTCAGAAGATG (SEQ ID  4 0.4 uMNO: 24) (synthesized by Beijing Liuhe BGI) NF H₂O 17

After mixing, it was placed in a PCR machine, the following program wasset: 95° C. for 3 min, 11 cycles (98° C. for 20 s, 58° C. for 20 s, 72°C. for 3 min), 72° C. for 5 min, 4° C. for co. After the reaction wascompleted, XP beads were used to purify and recover. The dsDNAconcentration was quantified using the qubit kit.

3. Sequencing. 80 fmol of the amplification product after the aboveinterruption was taken to prepare DNB. 40 ul of the following reactionsystem was prepared:

Ingredient Volume (ul) Final concentrationAmplification product after the above 80 fmol (X) interruption10x phi29 buffer (produced by BGI)  4 1X DNB primer sequence 10 uM  41 uM (CGAGAACGTCTCCGTAGCCATGTC, SEQ ID NO: 25)(synthesized by Beijing Liuhe BGI) H₂O 32-x

The above reaction system was placed in a PCR machine for reaction, andthe reaction conditions were as follows: 95° C. for 3 min, 40° C. for 3min After the reaction, it was placed on ice, added with 40 ul of mixedenzyme I and 2 ul of mixed enzyme II required to prepare DNB in DNBSEQsequencing kit, as well as 1 ul of ATP (100 mM mother liquor, ThermoFisher) and 0.1 ul of T4 ligase (produced by BGI). After mixing well,the above reaction system was transferred to a PCR machine at 30° C. andreacted for 20 minutes to form DNB. The DNB was loaded to the sequencingchip of MGISEQ2000 according to the method described in the PESO kit ofMGISEQ2000, and the sequencing was performed according to the relevantinstructions with the customer sequencing mode, wherein the sequencingof first strand was divided into two stages, i.e., sequencing 20 bp andthen sequencing 10 bp probe tag sequence, and 50 bp was sequenced forsecond strand.

Data Analysis

1. The 20 bp sequence of first strand obtained by cDNA sequencing wasmatched with the fq of spatial information sequence on the chip (thesequencing result obtained in step 3 in Example 4) by alignment. Thematching result was shown in FIG. 11 , in which the bright arearepresented the region where the 20 bp of cDNA sequencing exactlymatched the capture chip, and this region represented the region on thecapture chip for tissue capture. It showed that the capture chip coulduse the spatial positioning region to accurately locate the tissuecapture region.

2. The DNB matched the capture chip by cDNA sequencing was furtheranalyzed, and the alignment analysis between the second strandsequencing results of cDNA (mRNA expression in reaction tissue) of theseDNB reads and mouse genome was performed. For the DNB aligned to themouse genome, the mouse mRNA information was aligned to the capture chipthrough the 20 bp sequencing result. As shown in FIG. 12 , the left sideshowed the overall picture of the mRNA expression in the analyzed tissuesection, and the overall picture showed that this capture chip couldanalyze mRNA expression differences in tissues; the right side of thisfigure showed the tissue expression level of a randomly selected geneexpressed in mouse cerebellum, which indicated that this chip couldanalyze the expression differences of a certain gene in the wholetissue.

Although the specific embodiments of the present invention have beendescribed in detail, those skilled in the art will understand thatvarious modifications and changes can be made to the details accordingto all the teachings that have been published, and these changes arewithin the protection scope of the present invention. All of the presentinvention is given by the appended claims and any equivalents thereof.

1. A nucleic acid array for detecting spatial information of a nucleicacid in a sample, which comprises a solid support (e.g., a chip) withmultiple kinds of carrier sequences attached to its surface, in whicheach kind of carrier sequence occupies a different position in thearray, said each kind of carrier sequence comprises a plurality ofcopies of the carrier sequence, and the carrier sequence in thedirection from 5′ to 3′ comprises a positioning sequence and a firstimmobilization sequence, wherein, the positioning sequence has a uniquenucleotide sequence corresponding to the position of the kind of carriersequence on the array; the first immobilization sequence allowsannealing to its complementary nucleotide sequence and initiating anextension reaction.
 2. The nucleic acid array according to claim 1,wherein the nucleic acid array further comprises a first nucleic acidmolecule, the first nucleic acid molecule in the direction from 5′ to 3′comprises: a complement of the first immobilization sequence and acomplement of the positioning sequence, and the first nucleic acidmolecule hybridizes to the first immobilization sequence and thepositioning sequence of the carrier sequence to form a double strand;preferably, each copy of each kind of carrier sequence comprises a firstnucleic acid molecule hybridized therewith.
 3. The nucleic acid arrayaccording to claim 1 or 2, wherein the nucleic acid array furthercomprises a second nucleic acid molecule, the second nucleic acidmolecule is ligated to the first nucleic acid molecule, and the secondnucleic acid molecule comprises a capture sequence; the capture sequenceis capable of hybridizing with the whole or part of the nucleic acid tobe captured, and comprises: (a) an oligonucleotide sequence capable ofcapturing mRNA; and/or, (b) a random or degenerate oligonucleotidesequence; or, (c) a specific sequence for a specific target nucleicacid; and, the capture sequence has a free 3′ end to enable the secondnucleic acid molecule to function as an extension primer; preferably,each first nucleic acid molecule is ligated to the second nucleic acidmolecule.
 4. The nucleic acid array according to claim 1 or 2, whereineach carrier sequence further comprises a second immobilization sequenceat its 5′ end, and the second immobilization sequence allows annealingto its complementary nucleotide sequence; the second immobilizationsequence allows annealing to its complementary nucleotide sequence andinitiating an extension reaction.
 5. The nucleic acid array according toclaim 4, wherein the nucleic acid array further comprises a secondnucleic acid molecule, and the second nucleic acid molecule in thedirection from 5′ to 3′ comprises a complement of the secondimmobilization sequence and a capture sequence; the complement of secondimmobilization sequence hybridizes to the second immobilization sequenceof the carrier sequence to form a double strand; the capture sequence iscapable of hybridizing with the whole or part of the nucleic acid to becaptured, and comprises: (a) an oligonucleotide sequence capable ofcapturing mRNA; and/or, (b) a random or degenerate oligonucleotidesequence; or, (c) a specific sequence for a specific target nucleicacid; and, the capture sequence has a free 3′ end to enable the secondnucleic acid molecule to function as an extension primer; preferably,each copy of each kind of carrier sequence comprises a second nucleicacid molecule hybridized therewith.
 6. The nucleic acid array accordingto claim 1, wherein the multiple copies of carrier sequence are anamplification product formed by amplification using a complementarysequence of the carrier sequence as a template, and the amplification isselected from rolling circle amplification (RCA), bridge PCRamplification, multiple strand displacement amplification (MDA) oremulsion PCR amplification; preferably, the multiple copies of carriersequence are a DNB formed by a concatemer of the carrier sequence;preferably, the multiple copies of carrier sequence are a DNB formed byrolling circle amplification using a complementary sequence of thecarrier sequence as a template; preferably, the multiple copies ofcarrier sequence are a DNA cluster formed by a clone population of thecarrier sequence; for example, the multiple copies of carrier sequenceare a DNA cluster formed by bridge PCR amplification using acomplementary sequence of the carrier sequence as a template; forexample, the multiple copies of carrier sequence are a DNA clusterformed by emulsion PCR amplification using a complementary sequence ofthe carrier sequence as a template; for example, the multiple copies ofcarrier sequence are a DNA cluster formed by multiple stranddisplacement amplification using a complementary sequence of the carriersequence as a template.
 7. The nucleic acid array according to claim 2,wherein the first nucleic acid molecule further comprises a uniquemolecular identifier (UMI) sequence, and the UMI sequence is located atthe 5′ end of the complement of first immobilization sequence; the UMIsequence is a nucleotide sequence consisting of at least 1 (for example,at least 2, at least 3, at least 4, or at least 5; for example, 5 to100) nucleotide N, each N is independently any one of A, C, G and T;preferably, the UMI sequence contained in each first nucleic acidmolecule is different from each other.
 8. The nucleic acid arrayaccording to claim 3, wherein the second nucleic acid molecule furthercomprises a UMI sequence, and the UMI sequence is located at the 5′ endof the capture sequence; the UMI sequence is a nucleotide sequenceconsisting of at least 1 (for example, at least 2, at least 3, at least4, or at least 5; for example, 5 to 100) nucleotide N, each N isindependently any one of A, C, G and T; preferably, the UMI sequencecontained in each second nucleic acid molecule is different from eachother.
 9. The nucleic acid array according to claim 1, wherein the solidsupport is a chip; preferably, the solid support can be used as asequencing platform, such as a sequencing chip; preferably, the solidsupport is a high-throughput sequencing chip, such as a high-throughputsequencing chip used in Illumina, MGI or Thermo Fisher sequencingplatform.
 10. (canceled)
 11. The nucleic acid array according to claim1, wherein the carrier sequence further comprises a capture sequencetemplate located upstream of the positioning sequence, the capturesequence template comprises a complementary sequence of the capturesequence, and the capture sequence is capable of hybridizing with thewhole or part of the nucleic acid to be captured, which comprises: (a)an oligonucleotide sequence capable of capturing mRNA; and/or, (b) arandom or degenerate oligonucleotide sequence; or, (c) a specificsequence for a specific target nucleic acid; and, the firstimmobilization sequence of the carrier sequence also comprises acleavage site, and the cleavage can be selected from enzymatic cleavagewith nicking enzyme, enzymatic cleavage with USER enzyme, photocleavage,chemical cleavage or CRISPR-based cleavage; and, the nucleic acid arrayfurther comprises a first nucleic acid molecule, and the first nucleicacid molecule in the direction from 5′ to 3′ comprises: a bindingregion, a cleavage region, and a carrier sequence complementary region,the binding region comprises a linker capable of ligating to the surfaceof the solid support; the cleavage region comprises a cleavage site; thecarrier sequence complementary region comprises a sequence that can becomplementary to the carrier sequence, and in the direction from 5′ to3′, comprises: a complement of the first immobilization sequence, acomplement of the positioning sequence, and a capture sequence; and, thecapture sequence has a free 3′ end to enable the first nucleic acidmolecule to function as an extension primer; and, the carrier sequencecomplementary region of the first nucleic acid molecule hybridizes tothe carrier sequence to form a double strand; preferably, each copy ofeach kind of carrier sequence comprises a first nucleic acid moleculehybridized therewith.
 12. The nucleic acid array according to claim 11,wherein the carrier sequence further comprises a complement of UMIsequence located downstream of the capture sequence template andupstream of the first immobilization sequence, the complement of UMIsequence is complementary to the UMI sequence, the UMI sequence is anucleotide sequence consisting of at least 1 (for example, at least 2,at least 3, at least 4, or at least 5; for example, 5 to 100) nucleotideN, and each N is independently any one of A, C, G and T; and, thecarrier sequence complementary region of the first nucleic acid moleculefurther comprises the UMI sequence located upstream of the capturesequence and downstream of the complement of first immobilizationsequence; preferably, the complement of UMI sequence is located betweenthe positioning sequence and the capture sequence template, or betweenthe first immobilization sequence and the positioning sequence;preferably, each copy of each kind of carrier sequence (i.e., carriersequences comprising the same positioning sequence) comprises acomplement of UMI sequence different from each other.
 13. The nucleicacid array according to claim 11, wherein the linker of the firstnucleic acid molecule is a linking group capable of coupling with anactivating group (e.g., NH₂), and the surface of the solid support ismodified by the activating group (e.g., NH₂); preferably, the linkercomprises —SH, -DBCO or —NHS; preferably, the linker is

 (Azido-dPEG®8-NHS ester) is attached to the surface of the solidsupport.
 14. The nucleic acid array according to claim 11, wherein thecleavage site contained in the cleavage region of the first nucleic acidmolecule is a site where controlled cleavage can be performed by achemical, enzymatic, or photochemical method; preferably, the cleavagesite contained in the cleavage region of the first nucleic acid moleculeis an enzyme cleavage site; preferably, the cleavage region of the firstnucleic acid molecule is different from the cleavage site contained inthe first immobilization sequence of the carrier sequence.
 15. Thenucleic acid array according to claim 11, wherein the solid support is achip; preferably, the solid support can be used as a sequencingplatform, such as a sequencing chip; preferably, the solid support is ahigh-throughput sequencing chip, such as a high-throughput sequencingchip used in Illumina, MGI or Thermo Fisher sequencing platform. 16.(canceled)
 17. A kit, which comprises: (i) the nucleic acid arrayaccording to claim 2, wherein the nucleic acid array does not comprise asecond nucleic acid molecule; and, (ii) a second nucleic acid molecule,wherein the second nucleic acid molecule in the direction from 5′ to 3′comprises an immobilization region and a capture sequence; the capturesequence is capable of hybridizing with the whole or part of the nucleicacid to be captured, and comprises: (a) an oligonucleotide sequencecapable of capturing mRNA; and/or, (b) a random or degenerateoligonucleotide sequence; or, (c) a specific sequence for a specifictarget nucleic acid; and, the capture sequence has a free 3′ end toenable the second nucleic acid molecule to function as an extensionprimer.
 18. The kit according to claim 17, wherein the immobilizationregion of the second nucleic acid molecule comprises a double-strandednucleic acid sequence (for example, a double-stranded DNA sequence). 19.A kit comprising: (i) the nucleic acid array according to claim 4, and(ii) a second nucleic acid molecule, wherein the second nucleic acidmolecule in the direction from 5′ to 3′ comprises an immobilizationregion and a capture sequence; the capture sequence is capable ofhybridizing with the whole or part of the nucleic acid to be captured,and comprises: (a) an oligonucleotide sequence capable of capturingmRNA; and/or, (b) a random or degenerate oligonucleotide sequence; or,(c) a specific sequence for a specific target nucleic acid; and, thecapture sequence has a free 3′ end to enable the second nucleic acidmolecule to function as an extension primer; wherein the immobilizationregion of the second nucleic acid molecule comprises a complement ofsecond immobilization sequence.
 20. The kit according to claim 17,wherein the first nucleic acid molecule further comprises a uniquemolecular identifier (UMI) sequence, and the UMI sequence is located atthe 5′ end of the complement of first immobilization sequence; or, thesecond nucleic acid molecule further comprises a UMI sequence, and theUMI sequence is located at the 5′ end of the capture sequence; the UMIsequence is a nucleotide sequence consisting of at least 1 (for example,at least 2, at least 3, at least 4, or at least 5; for example, 5 to100) nucleotide N, each N is independently any one of A, C, G and T. 21.A method for generating a nucleic acid array for detecting spatialinformation of a nucleic acid in a biological sample, which comprisesthe following steps: (1) providing multiple kinds of carrier sequences,each kind of carrier sequence comprises a plurality of copies of thecarrier sequence, and the carrier sequence in the direction from 5′ to3′ comprises a positioning sequence and a first immobilization sequence,the positioning sequence has a unique nucleotide sequence correspondingto the position of the kind of carrier sequence on the array; the firstimmobilization sequence allows annealing to its complementary nucleotidesequence and initiating an extension reaction; (2) ligating the multiplekinds of carrier sequences to a surface of a solid support (e.g., achip); (3) providing a first primer, and perform a primer extensionreaction by using the carrier sequence as a template, so that a regionof the first immobilization sequence and the positioning sequence of thecarrier sequence forms a double strand, wherein the strand thathybridizes to the carrier sequence is a first nucleic acid molecule, thefirst nucleic acid molecule in the direction from 5′ to 3′ comprises thefirst immobilization sequence and a complementary sequence of thepositioning sequence; wherein, the first primer comprises a firstimmobilization sequence complementary region at its 3′ end, the firstimmobilization sequence complementary region comprises a complementarysequence of the first immobilization sequence or a fragment thereof, andhas a free 3′ end.
 22. The method according to claim 21, wherein in step(1), the multiple kinds of carrier sequences are provided by thefollowing steps: (i) providing multiple kinds of carrier sequencetemplates, the carrier sequence template comprising a complementarysequence of the carrier sequence; (ii) perform a nucleic acidamplification reaction by using each kind of carrier sequence templateas a template, to obtain an amplification product of each kind ofcarrier sequence template, the amplification product comprising aplurality of copies of the carrier sequence; preferably, theamplification is selected from rolling circle amplification (RCA),bridge PCR amplification, multiple strand displacement amplification(MDA) or emulsion PCR amplification; preferably, rolling circleamplification is performed to obtain a DNB formed by a concatemer of thecarrier sequence; preferably, bridge PCR amplification, emulsion PCRamplification or multiple strand displacement amplification is performedto obtain a DNA cluster formed by a clone population of the carriersequence.
 23. The method according to claim 21 or 22, wherein the methodfurther comprises the following steps: (4) providing a second nucleicacid molecule, the second nucleic acid molecule comprising a capturesequence; the capture sequence is capable of hybridizing with the wholeor part of the nucleic acid to be captured, which comprises: (a) anoligonucleotide sequence capable of capturing mRNA; and/or, (b) a randomor degenerate oligonucleotide sequence; or, (c) a specific sequence fora specific target nucleic acid; and, the capture sequence has a free 3′end to enable the second nucleic acid molecule to function as anextension primer, (5) ligating the second nucleic acid molecule to thefirst nucleic acid molecule (for example, using a ligase to ligate thesecond nucleic acid molecule to the first nucleic acid molecule). 24.The method according to claim 23, wherein the second nucleic acidmolecule in the direction from 5′ to 3′ comprises an immobilizationregion and a capture sequence, and the immobilization region comprises adouble-stranded DNA sequence.
 25. The method according to claim 23,wherein each carrier sequence further comprises a second immobilizationsequence at its 5′ end, and the second immobilization sequence allowsannealing to its complementary nucleotide sequence; the method furthercomprises the following steps: (4) providing a second nucleic acidmolecule, the second nucleic acid molecule in the direction from 5′ to3′ comprising a complement of second immobilization sequence and acapture sequence; the complement of second immobilization sequenceallows hybridization to its complementary nucleotide sequence; thecapture sequence is capable of hybridizing with the whole or part of thenucleic acid to be captured, and comprises: (a) an oligonucleotidesequence capable of capturing mRNA; and/or, (b) a random or degenerateoligonucleotide sequence; or, (c) a specific sequence for a specifictarget nucleic acid; and, the capture sequence has a free 3′ end toenable the second nucleic acid molecule to function as an extensionprimer; (5) hybridizing the complement of second immobilization sequencewith the second immobilization sequence under a condition that allowannealing, thereby ligating the second nucleic acid molecule to thecarrier sequence; (6) optionally, ligating the first nucleic acidmolecule and the second nucleic acid molecule that are hybridized to thecarrier sequence respectively (for example, using a ligase to ligate thesecond nucleic acid molecule to the first nucleic acid molecule). 26.The method according to claim 23, wherein, in step (3), the first primerfurther comprises a unique molecular identifier (UMI) at the 5′ end ofits first immobilization sequence complementary region, so that thefirst nucleic acid molecule comprises the UMI sequence at the 5′ end ofits complement of first immobilization sequence; or, in step (4), thesecond nucleic acid molecule further comprises a UMI sequence, and theUMI sequence is located at the 5′ end of the capture sequence; the UMIsequence is a nucleotide sequence consisting of at least 1 (for example,at least 2, at least 3, at least 4, or at least 5; for example, 5 to100) nucleotide N, each N is independently any one of A, C, G and T. 27.The method according to claim 21, wherein: in step (1), the carriersequence further comprises a capture sequence template located upstreamof the positioning sequence, the capture sequence template comprises acomplementary sequence of the capture sequence, and the capture sequencecan hybridize to the whole or part of the nucleic acid to be captured,which comprises: (a) an oligonucleotide sequence capable of capturingmRNA; and/or, (b) a random or degenerate oligonucleotide sequence; or,(c) a specific sequence for a specific target nucleic acid; and thefirst immobilization sequence of the carrier sequence also comprises acleavage site, and the cleavage can be selected from enzymatic cleavagewith nicking enzyme, enzymatic cleavage with USER enzyme, photocleavage,chemical cleavage or CRISPR-based cleavage; in step (3), a region of thefirst immobilization sequence, the positioning sequence and the capturesequence template of the carrier sequence forms a double strand, so thatthe first nucleic acid molecule in the direction from 5′ to 3′ comprisesa complement of first immobilization sequence, a complement ofpositioning sequence and a capture sequence; wherein, the first primerin the direction from 5′ to 3′ comprises a binding region, an cleavageregion, and a first immobilization sequence complementary region, thebinding region comprises a linker capable of ligating to the surface ofthe solid support, and the cleavage region comprises a cleavage site;and, the method further comprises the following steps: (4) ligating thefirst primer to the surface of the solid support; wherein, steps (3) and(4) are performed in any order; (5) optionally, performing cleavage atthe cleavage site contained in the first immobilization sequence of thecarrier sequence to digest the carrier sequence, so that the extensionproduct in step (3) is separated from the template where such extensionproduct is formed (i.e., carrier sequence), and the first nucleic acidmolecule is therefore ligated to the surface of the solid support (e.g.,chip); preferably, each kind of carrier sequence is a DNB formed by aconcatemer of the multiple copies of carrier sequence.
 28. The methodaccording to claim 27, wherein, in step (1), the cleavage site containedin the first immobilization sequence is a cleavage site of nickingenzyme; preferably, the nicking enzyme is selected from USER, BamHI, andBmtI.
 29. The method according to claim 27, wherein, in step (1), thecarrier sequence further comprises a complement of UMI sequence locateddownstream of the capture sequence template and upstream of the firstimmobilization sequence, the complement of UMI sequence is complementaryto a UMI sequence, and the UMI sequence is a nucleotide sequenceconsisting of at least 1 (for example, at least 2, at least 3, at least4, or at least 5; for example, 5 to 100) nucleotide N, each N isindependently any one of A, C, G and T; and, in step (3), a region ofthe first immobilization sequence, the positioning sequence, the capturesequence template, and the complement of UMI sequence of the carriersequence forms a double strand, so that the first nucleic acid moleculein the direction from 5′ to 3′ comprises the complement of firstimmobilization sequence, the complement of positioning sequence and thecapture sequence, and the UMI sequence located upstream of the capturesequence and downstream of the complement of first immobilizationsequence; preferably, the complement of UMI sequence is located betweenthe positioning sequence and the capture sequence template, or betweenthe first immobilization sequence and the positioning sequence.
 30. Themethod according to claim 27, wherein the linker of the first primer isa linking group capable of coupling with an activating group (e.g.,NH₂), and the surface of the solid support is modified by the activatinggroup (e.g., NH₂); preferably, the linker comprises —SH, -DBCO or —NHS;preferably, the linker is

 (Azido-dPEG®8-NHS ester) is attached to the surface of the solidsupport.
 31. The method according to claim 27, wherein the cleavage sitecontained in the cleavage region of the first primer is a site wherecontrolled cleavage can be performed by a chemical, enzymatic, orphotochemical method; preferably, the cleavage site contained in thecleavage region of the first primer is an enzyme cleavage site;preferably, the cleavage region of the first primer is different fromthe cleavage site contained in the first immobilization sequence of thecarrier sequence.
 32. (canceled)
 33. The method according to claim 21,wherein the solid support is a chip; preferably, the solid support canbe used as a sequencing platform, such as a sequencing chip; preferably,the solid support is a high-throughput sequencing chip, such as ahigh-throughput sequencing chip used in Illumina, MGI or Thermo Fishersequencing platform.
 34. The method according to claim 21, wherein, instep (3), while performing an extension reaction, the carrier sequenceis sequenced to obtain the sequence information of the positioningsequence contained in the carrier sequence.
 35. The method according toclaim 21, wherein, before step (3), a step of sequencing the carriersequence is comprised; preferably, after the sequencing is completed,washing is performed to remove dNTP added to the synthetic strand due tothe sequencing. 36-37. (canceled)
 38. A method for detecting spatialinformation of a nucleic acid in a sample, which comprises the followingsteps: (1) providing the nucleic acid array according to claim 3;wherein, the nucleic acid array comprises multiple kinds of carriersequences attached to a surface of a solid support (e.g., a chip), eachkind of carrier sequence occupies a different position in the array, andsaid each kind of carrier sequence comprises a plurality of copies ofthe carrier sequence; each carrier sequence comprises a first nucleicacid molecule hybridized therewith, and the first nucleic acid moleculeis ligated to a second nucleic acid molecule; the first nucleic acidmolecule comprises a complement of positioning sequence corresponding tothe position of the kind of carrier sequence on the array, the secondnucleic acid molecule comprises a capture sequence capable of capturingthe nucleic acid in the sample; (2) contacting the nucleic acid arraywith the sample to be tested under a condition that allows annealing, sothat the nucleic acid in the sample to be tested anneals to the capturesequence of the second nucleic acid molecule, and thus the position ofthe nucleic acid can be correlated with the position of the carriersequence on the nucleic acid array; (3) performing a primer extensionreaction by using the ligated first and second nucleic acid molecules asa primer, and using the captured nucleic acid molecule as a templateunder a condition that allows the primer extension, to produce anextension product, in which the strand that hybridizes to the capturednucleic acid molecule has the complement of positioning sequencecontained in the first nucleic acid molecule as a spatial informationtag; and/or, performing a primer extension reaction by using thecaptured nucleic acid molecule as a primer, and using the ligated firstand second connected nucleic acid molecules as a template under acondition that allows the primer extension, to produce an extendedcaptured nucleic acid molecule, in which the extended captured nucleicacid molecule has the positioning sequence as a spatial information tag;(4) releasing at least part of the nucleic acid molecules labeled withspatial information tags from the surface of the array, wherein the partcomprises the positioning sequence or its complementary strand and thecaptured nucleic acid molecule or its complementary strand; and (5)directly or indirectly analyzing the sequence of the nucleic acidmolecule released in step (4); preferably, the spatial information ofthe nucleic acid comprises the location, distribution and/or expressionof the nucleic acid; preferably, the sample is a tissue sample, such asa tissue section; preferably, the tissue section is prepared from afixed tissue, for example, a formalin-fixed paraffin-embedded (FFPE)tissue or deep-frozen tissue.
 39. The method according to claim 38,which is used to detect a transcriptome in a sample, wherein: (a) instep (3), generating a cDNA molecule from the captured RNA molecule byusing the ligated first and second nucleic acid molecules as a reversetranscription primer, the cDNA molecule has the complement ofpositioning sequence contained in the first nucleic acid molecule as aspatial information tag, and optionally, amplifying the cDNA molecule;and, (b) in step (4), releasing at least part of the cDNA moleculesand/or their amplicons from the surface of the array, wherein thereleased nucleic acid molecule may be the first and/or second strand ofthe cDNA molecule or an amplicon thereof, and wherein the part comprisesthe positioning sequence or its complementary strand; preferably, instep (1), the capture sequence comprises an oligonucleotide sequencecapable of capturing mRNA.
 40. A method for detecting spatialinformation of a nucleic acid in a sample, which comprises the followingsteps: (1) providing the nucleic acid array according to claim 11;wherein the nucleic acid array comprises a solid support (e.g., a chip)with multiple kinds of carrier sequences attached to its surface, eachkind of carrier sequence occupies a different position in the array, andsaid each kind of carrier sequence comprises a plurality of copies ofthe carrier sequence; each carrier sequence comprises a first nucleicacid molecule hybridized therewith, and the first nucleic acid moleculecomprises a complement of positioning sequence corresponding to theposition of the kind of carrier sequence on the array and a capturesequence capable of capturing the nucleic acid in the sample; (2)contacting the nucleic acid array with the sample to be tested under acondition that allows annealing, so that the nucleic acid in the sampleto be tested anneals to the capture sequence of the first nucleic acidmolecule, and thus the position of the nucleic acid can be correlatedwith the position of the first nucleic acid molecule on the nucleic acidarray; (3) performing a primer extension reaction by using the firstnucleic acid molecule as a primer and using the captured nucleic acidmolecule as a template under a condition that allows the primerextension, to produce an extension product, in which the strand thathybridizes to the captured nucleic acid molecule has the complement ofpositioning sequence contained in the first nucleic acid molecule as aspatial information tag; (4) releasing at least part of the nucleic acidmolecules labeled with the spatial information tags from the surface ofthe array, wherein the part comprises the positioning sequence or itscomplementary strand and the captured nucleic acid molecule or itscomplementary strand; and (5) directly or indirectly analyzing thesequence of the nucleic acid molecule released in step (4); preferably,the spatial information of the nucleic acid comprises the location,distribution and/or expression of the nucleic acid; preferably, thesample is a tissue sample, such as a tissue section; preferably, thetissue section is prepared from a fixed tissue, for example, aformalin-fixed paraffin-embedded (FFPE) tissue or deep-frozen tissue.41. The method according to claim 40, in which the method is used todetect a transcriptome in a sample, wherein: in step (3), generating acDNA molecule from the captured RNA molecule by using the first nucleicacid molecule as an RT primer, the cDNA molecule has the complement ofpositioning sequence contained in the first nucleic acid molecule as aspatial information tag, and optionally, amplifying the cDNA molecule;in step (4), releasing at least part of the cDNA molecules and/or theiramplicons from the surface of the array, wherein the released nucleicacid molecule may be the first and/or second strand of the cDNA moleculeor an amplicon thereof, and wherein the part comprises the positioningsequence or its complementary strand; preferably, in step (1), thecapture sequence comprises an oligonucleotide sequence capable ofcapturing mRNA. 42-49. (canceled)
 50. The nucleic acid array accordingto claim 5, wherein the second nucleic acid molecule further comprises aUMI sequence, and the UMI sequence is located at the 5′ end of thecapture sequence; the UMI sequence is a nucleotide sequence consistingof at least 1 (for example, at least 2, at least 3, at least 4, or atleast 5; for example, 5 to 100) nucleotide N, each N is independentlyany one of A, C, G and T; preferably, the UMI sequence contained in eachsecond nucleic acid molecule is different from each other.
 51. The kitaccording to claim 19, wherein the first nucleic acid molecule furthercomprises a unique molecular identifier (UMI) sequence, and the UMIsequence is located at the 5′ end of the complement of firstimmobilization sequence; or, the second nucleic acid molecule furthercomprises a UMI sequence, and the UMI sequence is located at the 5′ endof the capture sequence; the UMI sequence is a nucleotide sequenceconsisting of at least 1 (for example, at least 2, at least 3, at least4, or at least 5; for example, 5 to 100) nucleotide N, each N isindependently any one of A, C, G and T.
 52. A method for detectingspatial information of a nucleic acid in a sample, which comprises thefollowing steps: (1) providing the nucleic acid array according to claim5; wherein, the nucleic acid array comprises multiple kinds of carriersequences attached to a surface of a solid support (e.g., a chip), eachkind of carrier sequence occupies a different position in the array, andsaid each kind of carrier sequence comprises a plurality of copies ofthe carrier sequence; each carrier sequence comprises a first nucleicacid molecule and a second nucleic acid molecule that are hybridizedtherewith; the first nucleic acid molecule comprises a complement ofpositioning sequence corresponding to the position of the kind ofcarrier sequence on the array, the second nucleic acid moleculecomprises a capture sequence capable of capturing the nucleic acid inthe sample; (2) contacting the nucleic acid array with the sample to betested under a condition that allows annealing, so that the nucleic acidin the sample to be tested anneals to the capture sequence of the secondnucleic acid molecule, and thus the position of the nucleic acid can becorrelated with the position of the carrier sequence on the nucleic acidarray; (3) (i) when the first nucleic acid molecule and the secondnucleic acid molecule are not ligated to each other, ligating the firstnucleic acid molecule and the second nucleic acid molecule that arehybridized to each carrier sequence (for example, using a ligase);performing a primer extension reaction by using the ligated first andsecond nucleic acid molecules as a primer, and using the capturednucleic acid molecule as a template under a condition that allows theprimer extension, so as to produce an extension product, in which thestrand that hybridizes to the captured nucleic acid molecule has thecomplement of positioning sequence contained in the first nucleic acidmolecule as a spatial information tag; and/or, performing a primerextension reaction by using the captured nucleic acid molecule as aprimer, and using the ligated first and second nucleic acid molecules asa template under a condition that allows the primer extension, so as toproduce an extended captured nucleic acid molecule, in which theextended captured nucleic acid has the positioning sequence as a spatialinformation tag; alternatively, (ii) when the first nucleic acidmolecule and the second nucleic acid molecule are not ligated to eachother, performing a primer extension reaction by using the secondnucleic acid molecule as a primer and using the captured nucleic acidmolecule as a template under a condition that allows the primerextension, to produce an extended second nucleic acid molecule, in whichthe extended second nucleic acid molecule comprises a complementarysequence of the captured nucleic acid; ligating the first nucleic acidmolecule and the extended second nucleic acid molecule that arehybridized to each carrier sequence (for example, by using a ligase), inwhich the extended second nucleic acid molecule which is ligated to thefirst nucleic acid molecule has the complement of positioning containedin the first nucleic acid molecule as a spatial information tag; (4)releasing at least part of the nucleic acid molecules labeled withspatial information tags from the surface of the array, wherein the partcomprises the positioning sequence or its complementary strand and thecaptured nucleic acid molecule or its complementary strand; and (5)directly or indirectly analyzing the sequence of the nucleic acidmolecule released in step (4); preferably, the spatial information ofthe nucleic acid comprises the location, distribution and/or expressionof the nucleic acid; preferably, the sample is a tissue sample, such asa tissue section; preferably, the tissue section is prepared from afixed tissue, for example, a formalin-fixed paraffin-embedded (FFPE)tissue or deep-frozen tissue.
 53. The method according to claim 52,which is used to detect a transcriptome in a sample, wherein: (a) instep (3)(i), generating a cDNA molecule from the captured RNA moleculeby using the ligated first and second nucleic acid molecules as areverse transcription primer, the cDNA molecule has the complement ofpositioning sequence contained in the first nucleic acid molecule as aspatial information tag, and optionally, amplifying the cDNA molecule;or, in step (3)(ii), generating a cDNA molecule from the captured RNAmolecule by using the second nucleic acid molecule as a reversetranscription primer, ligating the first nucleic acid molecule and thecDNA molecule that hybridizes to each carrier sequence (for example, byusing ligase), to generate a cDNA molecule having the complement ofpositioning sequence contained in the first nucleic acid molecule as aspatial information tag, and optionally, amplifying the cDNA molecule;and, (b) in step (4), releasing at least part of the cDNA moleculesand/or their amplicons from the surface of the array, wherein thereleased nucleic acid molecule may be the first and/or second strand ofthe cDNA molecule or an amplicon thereof, and wherein the part comprisesthe positioning sequence or its complementary strand; preferably, instep (1), the capture sequence comprises an oligonucleotide sequencecapable of capturing mRNA.
 54. The method according to claim 38, whichis characterized by one or more of the following: (i) in step (1), themultiple copies of the carrier sequence is a DNB formed by a concatemerof the carrier sequence, or the multiple copies of the carrier sequenceis a DNA cluster formed by a clone population of the carrier sequence;(ii) in step (5), the sequence analysis comprises sequencing or asequence-specific PCR reaction; (iii) the method further comprises step(6): correlating the sequence analysis information obtained in step (5)to an image of the sample, wherein the sample is imaged before or afterstep (3); preferably, the imaging of the sample uses light, brightfield, dark field, phase contrast, fluorescence, reflection,interference, confocal microscopy or a combination thereof; (iv) beforeor after the nucleic acid molecule labeled with spatial information tagis released from the surface of the array, the complementary strand isgenerated; preferably, the synthesis of the complementary strand uses arandom primer and a strand displacement polymerase; (v) before thesequence analysis, the method further comprises a step of amplifying thenucleic acid molecule labeled with the spatial information tag;preferably, the amplification step is performed after the nucleic acidmolecule labeled with the spatial information tag is released from thearray, or the amplification step is performed in situ on the array;preferably, the amplification step comprises PCR; (vi) before thesequence analysis, the method further comprises a step of purifying thereleased nucleic acid molecule; (vii) before step (4), the methodfurther comprises a step of washing the array to remove a residue of thesample (e.g., tissue); (viii) in step (4), the nucleic acid molecule isreleased from the surface of the array by the following method: (i)nucleic acid cleavage; (ii) denaturation; and/or (iii) physical method.55. The method according to claim 52, which is characterized by one ormore of the following: (i) in step (1), the multiple copies of thecarrier sequence is a DNB formed by a concatemer of the carriersequence, or the multiple copies of the carrier sequence is a DNAcluster formed by a clone population of the carrier sequence; (ii) instep (5), the sequence analysis comprises sequencing or asequence-specific PCR reaction; (iii) the method further comprises step(6): correlating the sequence analysis information obtained in step (5)to an image of the sample, wherein the sample is imaged before or afterstep (3); preferably, the imaging of the sample uses light, brightfield, dark field, phase contrast, fluorescence, reflection,interference, confocal microscopy or a combination thereof; (iv) beforeor after the nucleic acid molecule labeled with spatial information tagis released from the surface of the array, the complementary strand isgenerated; preferably, the synthesis of the complementary strand uses arandom primer and a strand displacement polymerase; (v) before thesequence analysis, the method further comprises a step of amplifying thenucleic acid molecule labeled with the spatial information tag;preferably, the amplification step is performed after the nucleic acidmolecule labeled with the spatial information tag is released from thearray, or the amplification step is performed in situ on the array;preferably, the amplification step comprises PCR; (vi) before thesequence analysis, the method further comprises a step of purifying thereleased nucleic acid molecule; (vii) before step (4), the methodfurther comprises a step of washing the array to remove a residue of thesample (e.g., tissue); (viii) in step (4), the nucleic acid molecule isreleased from the surface of the array by the following method: (i)nucleic acid cleavage; (ii) denaturation; and/or (iii) physical method.56. The method according to claim 40, which is characterized by one ormore of the following: (i) in step (1), the multiple copies of thecarrier sequence is a DNB formed by a concatemer of the carriersequence, or the multiple copies of the carrier sequence is a DNAcluster formed by a clone population of the carrier sequence; (ii) instep (5), the sequence analysis comprises sequencing or asequence-specific PCR reaction; (iii) the method further comprises step(6): correlating the sequence analysis information obtained in step (5)to an image of the sample, wherein the sample is imaged before or afterstep (3); preferably, the imaging of the sample uses light, brightfield, dark field, phase contrast, fluorescence, reflection,interference, confocal microscopy or a combination thereof; (iv) beforeor after the nucleic acid molecule labeled with spatial information tagis released from the surface of the array, the complementary strand isgenerated; preferably, the synthesis of the complementary strand uses arandom primer and a strand displacement polymerase; (v) before thesequence analysis, the method further comprises a step of amplifying thenucleic acid molecule labeled with the spatial information tag;preferably, the amplification step is performed after the nucleic acidmolecule labeled with the spatial information tag is released from thearray, or the amplification step is performed in situ on the array;preferably, the amplification step comprises PCR; (vi) before thesequence analysis, the method further comprises a step of purifying thereleased nucleic acid molecule; (vii) before step (4), the methodfurther comprises a step of washing the array to remove a residue of thesample (e.g., tissue); (viii) in step (4), the nucleic acid molecule isreleased from the surface of the array by the following method: (i)nucleic acid cleavage; (ii) denaturation; and/or (iii) physical method.